Two new competing pathways establish the threshold for cyclin-B-Cdk1 activation at the meiotic G2/M transition.

PubMed

Hiraoka, Daisaku; Aono, Ryota; Hanada, Shin-Ichiro; Okumura, Eiichi; Kishimoto, Takeo

2016-08-15

Extracellular ligands control biological phenomena. Cells distinguish physiological stimuli from weak noise stimuli by establishing a ligand-concentration threshold. Hormonal control of the meiotic G2/M transition in oocytes is essential for reproduction. However, the mechanism for threshold establishment is unclear. In starfish oocytes, maturation-inducing hormones activate the PI3K-Akt pathway through the Gβγ complex of heterotrimeric G-proteins. Akt directly phosphorylates both Cdc25 phosphatase and Myt1 kinase, resulting in activation of cyclin-B-Cdk1, which then induces meiotic G2/M transition. Here, we show that cyclin-B-Cdk1 is partially activated after subthreshold hormonal stimuli, but this triggers negative feedback, resulting in dephosphorylation of Akt sites on Cdc25 and Myt1, thereby canceling the signal. We also identified phosphatase activity towards Akt substrates that exists independent of stimuli. In contrast to these negative regulatory activities, an atypical Gβγ-dependent pathway enhances PI3K-Akt-dependent phosphorylation. Based on these findings, we propose a model for threshold establishment in which hormonal dose-dependent competition between these new pathways establishes a threshold; the atypical Gβγ-pathway becomes predominant over Cdk-dependent negative feedback when the stimulus exceeds this threshold. Our findings provide a regulatory connection between cell cycle and signal transduction machineries. © 2016. Published by The Company of Biologists Ltd.

Ca2+ -stimulated adenylyl cyclases regulate ERK-dependent activation of MSK1 during fear conditioning.

PubMed

Sindreu, Carlos Balet; Scheiner, Zachary S; Storm, Daniel R

2007-01-04

The cAMP and ERK/MAP kinase (MAPK) signal transduction pathways are critical for hippocampus-dependent memory, a process that depends on CREB-mediated transcription. However, the extent of crosstalk between these pathways and the downstream CREB kinase activated during memory formation has not been elucidated. Here we report that PKA, MAPK, and MSK1, a CREB kinase, are coactivated in a subset of hippocampal CA1 pyramidal neurons following contextual fear conditioning. Activation of PKA, MAPK, MSK1, and CREB is absolutely dependent on Ca(2+)-stimulated adenylyl cyclase activity. We conclude that adenylyl cyclase activity supports the activation of MAPK, and that MSK1 is the major CREB kinase activated during training for contextual memory.

GLP-1 mediates antiapoptotic effect by phosphorylating Bad through a beta-arrestin 1-mediated ERK1/2 activation in pancreatic beta-cells.

PubMed

Quoyer, Julie; Longuet, Christine; Broca, Christophe; Linck, Nathalie; Costes, Safia; Varin, Elodie; Bockaert, Joël; Bertrand, Gyslaine; Dalle, Stéphane

2010-01-15

Strategies based on activating GLP-1 receptor (GLP-1R) are intensively developed for the treatment of type 2 diabetes. The exhaustive knowledge of the signaling pathways linked to activated GLP-1R within the beta-cells is of major importance. In beta-cells, GLP-1 activates the ERK1/2 cascade by diverse pathways dependent on either Galpha(s)/cAMP/cAMP-dependent protein kinase (PKA) or beta-arrestin 1, a scaffold protein. Using pharmacological inhibitors, beta-arrestin 1 small interfering RNA, and islets isolated from beta-arrestin 1 knock-out mice, we demonstrate that GLP-1 stimulates ERK1/2 by two temporally distinct pathways. The PKA-dependent pathway mediates rapid and transient ERK1/2 phosphorylation that leads to nuclear translocation of the activated kinases. In contrast, the beta-arrestin 1-dependent pathway produces a late ERK1/2 activity that is restricted to the beta-cell cytoplasm. We further observe that GLP-1 phosphorylates the cytoplasmic proapoptotic protein Bad at Ser-112 but not at Ser-155. We find that the beta-arrestin 1-dependent ERK1/2 activation engaged by GLP-1 mediates the Ser-112 phosphorylation of Bad, through p90RSK activation, allowing the association of Bad with the scaffold protein 14-3-3, leading to its inactivation. beta-Arrestin 1 is further found to mediate the antiapoptotic effect of GLP-1 in beta-cells through the ERK1/2-p90RSK-phosphorylation of Bad. This new regulatory mechanism engaged by activated GLP-1R involving a beta-arrestin 1-dependent spatiotemporal regulation of the ERK1/2-p90RSK activity is now suspected to participate in the protection of beta-cells against apoptosis. Such signaling mechanism may serve as a prototype to generate new therapeutic GLP-1R ligands.

Time-dependent LXR/RXR pathway modulation characterizes capillary remodeling in inflammatory corneal neovascularization.

PubMed

Mukwaya, Anthony; Lennikov, Anton; Xeroudaki, Maria; Mirabelli, Pierfrancesco; Lachota, Mieszko; Jensen, Lasse; Peebo, Beatrice; Lagali, Neil

2018-05-01

Inflammation in the normally immune-privileged cornea can initiate a pathologic angiogenic response causing vision-threatening corneal neovascularization. Inflammatory pathways, however, are numerous, complex and are activated in a time-dependent manner. Effective resolution of inflammation and associated angiogenesis in the cornea requires knowledge of these pathways and their time dependence, which has, to date, remained largely unexplored. Here, using a model of endogenous resolution of inflammation-induced corneal angiogenesis, we investigate the time dependence of inflammatory genes in effecting capillary regression and the return of corneal transparency. Endogenous capillary regression was characterized by a progressive thinning and remodeling of angiogenic capillaries and inflammatory cell retreat in vivo in the rat cornea. By whole-genome longitudinal microarray analysis, early suppression of VEGF ligand-receptor signaling and inflammatory pathways preceded an unexpected later-phase preferential activation of LXR/RXR, PPARα/RXRα and STAT3 canonical pathways, with a concurrent attenuation of LPS/IL-1 inhibition of RXR function and Wnt/β-catenin signaling pathways. Potent downstream inflammatory cytokines such as Cxcl5, IL-1β, IL-6 and Ccl2 were concomitantly downregulated during the remodeling phase. Upstream regulators of the inflammatory pathways included Socs3, Sparc and ApoE. A complex and coordinated time-dependent interplay between pro- and anti-inflammatory signaling pathways highlights a potential anti-inflammatory role of LXR/RXR, PPARα/RXRα and STAT3 signaling pathways in resolving inflammatory corneal angiogenesis.

Degradation pathway of 2-chloroethanol in Pseudomonas stutzeri strain JJ under denitrifying conditions.

PubMed

Dijk, John A; Gerritse, Jan; Schraa, Gosse; Stams, Alfons J M

2004-12-01

The pathway of 2-chloroethanol degradation in the denitrifying Pseudomonas stutzeri strain JJ was investigated. In cell-free extracts, activities of a phenazine methosulfate (PMS)-dependent chloroethanol dehydrogenase, an NAD-dependent chloroacetaldehyde dehydrogenase, and a chloroacetate dehalogenase were detected. This suggested that the 2-chloroethanol degradation pathway in this denitrifying strain is the same as found in aerobic bacteria that degrade chloroethanol. Activity towards primary alcohols, secondary alcohols, diols, and other chlorinated alcohols could be measured in cell-free extracts with chloroethanol dehydrogenase (CE-DH) activity. PMS and phenazine ethosulfate (PES) were used as primary electron acceptors, but not NAD, NADP or ferricyanide. Cells of strain JJ cultured in a continuous culture under nitrate limitation exhibited chloroethanol dehydrogenase activity that was a 12 times higher than in cells grown in batch culture. However, under chloroethanol-limiting conditions, CE-DH activity was in the same range as in batch culture. Cells grown on ethanol did not exhibit CE-DH activity. Instead, NAD-dependent ethanol dehydrogenase (E-DH) activity and PMS-dependent E-DH activity were detected.

Gtr/Ego-independent TORC1 activation is achieved through a glutamine-sensitive interaction with Pib2 on the vacuolar membrane.

PubMed

Ukai, Hirofumi; Araki, Yasuhiro; Kira, Shintaro; Oikawa, Yu; May, Alexander I; Noda, Takeshi

2018-04-01

TORC1 is a central regulator of cell growth in response to amino acids. The role of the evolutionarily conserved Gtr/Rag pathway in the regulation of TORC1 is well-established. Recent genetic studies suggest that an additional regulatory pathway, depending on the activity of Pib2, plays a role in TORC1 activation independently of the Gtr/Rag pathway. However, the interplay between the Pib2 pathway and the Gtr/Rag pathway remains unclear. In this study, we show that Pib2 and Gtr/Ego form distinct complexes with TORC1 in a mutually exclusive manner, implying dedicated functional relationships between TORC1 and Pib2 or Gtr/Rag in response to specific amino acids. Furthermore, simultaneous depletion of Pib2 and the Gtr/Ego system abolishes TORC1 activity and completely compromises the vacuolar localization of TORC1. Thus, the amino acid-dependent activation of TORC1 is achieved through the Pib2 and Gtr/Ego pathways alone. Finally, we show that glutamine induces a dose-dependent increase in Pib2-TORC1 complex formation, and that glutamine binds directly to the Pib2 complex. These data provide strong preliminary evidence for Pib2 functioning as a putative glutamine sensor in the regulation of TORC1.

Gtr/Ego-independent TORC1 activation is achieved through a glutamine-sensitive interaction with Pib2 on the vacuolar membrane

PubMed Central

Ukai, Hirofumi; Araki, Yasuhiro; Kira, Shintaro; Oikawa, Yu; May, Alexander I.

2018-01-01

TORC1 is a central regulator of cell growth in response to amino acids. The role of the evolutionarily conserved Gtr/Rag pathway in the regulation of TORC1 is well-established. Recent genetic studies suggest that an additional regulatory pathway, depending on the activity of Pib2, plays a role in TORC1 activation independently of the Gtr/Rag pathway. However, the interplay between the Pib2 pathway and the Gtr/Rag pathway remains unclear. In this study, we show that Pib2 and Gtr/Ego form distinct complexes with TORC1 in a mutually exclusive manner, implying dedicated functional relationships between TORC1 and Pib2 or Gtr/Rag in response to specific amino acids. Furthermore, simultaneous depletion of Pib2 and the Gtr/Ego system abolishes TORC1 activity and completely compromises the vacuolar localization of TORC1. Thus, the amino acid-dependent activation of TORC1 is achieved through the Pib2 and Gtr/Ego pathways alone. Finally, we show that glutamine induces a dose-dependent increase in Pib2-TORC1 complex formation, and that glutamine binds directly to the Pib2 complex. These data provide strong preliminary evidence for Pib2 functioning as a putative glutamine sensor in the regulation of TORC1. PMID:29698392

Time-dependent activation of MAPK/Erk1/2 and Akt/GSK3 cascades: modulation by agomelatine.

PubMed

Musazzi, Laura; Seguini, Mara; Mallei, Alessandra; Treccani, Giulia; Pelizzari, Mariagrazia; Tornese, Paolo; Racagni, Giorgio; Tardito, Daniela

2014-10-21

The novel antidepressant agomelatine, a melatonergic MT1/MT2 agonist combined with 5-HT2c serotonin antagonist properties, showed antidepressant action in preclinical and clinical studies. There is a general agreement that the therapeutic action of antidepressants needs the activation of slow-onset adaptations in downstream signalling pathways finally regulating neuroplasticity. In the last several years, particular attention was given to cAMP-responsive element binding protein (CREB)-related pathways, since it was shown that chronic antidepressants increase CREB phosphorylation and transcriptional activity, through the activation of calcium/calmodulin-dependent (CaM) and mitogen activated protein kinase cascades (MAPK/Erk1/2). Aim of this work was to analyse possible effects of chronic agomelatine on time-dependent changes of different intracellular signalling pathways in hippocampus and prefrontal/frontal cortex of male rats. To this end, measurements were performed 1 h or 16 h after the last agomelatine or vehicle injection. We have found that in naïve rats chronic agomelatine, contrary to traditional antidepressants, did not increase CREB phosphorylation, but modulates the time-dependent regulation of MAPK/Erk1/2 and Akt/glycogen synthase kinase-3 (GSK-3) pathways. Our results suggest that the intracellular molecular mechanisms modulated by chronic agomelatine may be partly different from those of traditional antidepressants and involve the time-dependent regulation of MAPK/Erk1/2 and Akt/GSK-3 signalling pathways. This could exert a role in the antidepressant efficacy of the drug.

Activated Macrophages Destroy Intracellular Leishmania Major Amastigotes by an l-Arginine-Dependent Killing Mechanism

DTIC Science & Technology

1990-01-01

atom of L-arginine and a precursor of the nitrite measured, may disrupt Fe- dependent enzymatic pathways vital to the survival of amastigotes within...geneti- a precursor of the nitrite measured, may disrupt Fe- cally susceptible BALB/c mice. The exact role of IFN-1 in dependent enzymatic pathways vital...induces the heme - dependent activation of 0 6 ± 4 89 80 guanylate cyclase. with the subsequent stimulation of 0.01 8 ± 3 85 67 the secondary messenger

Distinct effects of thrombopoietin depending on a threshold level of activated Mpl in BaF-3 cells.

PubMed

Millot, Gaël A; Vainchenker, William; Duménil, Dominique; Svinarchuk, Fédor

2002-06-01

Thrombopoietin (TPO) plays a critical role in megakaryopoiesis through binding to its receptor Mpl. This involves activation of various intracellular signaling pathways, including phosphoinositide 3-kinase (PI3K) and the mitogen-activated protein kinase (MAPK) pathways. Their precise role in TPO-mediated proliferation, survival and differentiation is not fully understood. In the present study, we show that TPO induces different biological responses in Mpl-transduced BaF-3 cells, depending on the cell surface density of Mpl and the resulting activation level of signaling pathways. TPO mediates cell proliferation in cells expressing high levels of Mpl but only mediates survival without proliferation in cells expressing low levels of the receptor. By using the kinase inhibitors PD98059 and LY294002, we further showed that the activation level of the PI3K and MAPK p42/44 pathways is a determining factor for the proliferative effect. In cells expressing low levels of Mpl, the survival effect was strongly dependent on the activation level of the PI3K/AKT, but not the MAPK p42/44 pathway. Moreover, this effect was correlated with the phosphorylation level of BAD but not with the expression level of Bcl-X(L). However, PI3K pathway inhibition did not increase apoptosis when BaF-3 cells proliferated in response to TPO, indicating a compensating mechanism from other Mpl signaling pathways in this case.

Glucocorticoids activate the ATP-ubiquitin-dependent proteolytic system in skeletal muscle during fasting

NASA Technical Reports Server (NTRS)

Wing, S. S.; Goldberg, A. L.; Goldberger, A. L. (Principal Investigator)

1993-01-01

Glucocorticoids are essential for the increase in protein breakdown in skeletal muscle normally seen during fasting. To determine which proteolytic pathway(s) are activated upon fasting, leg muscles from fed and fasted normal rats were incubated under conditions that block or activate different proteolytic systems. After food deprivation (1 day), the nonlysosomal ATP-dependent process increased by 250%, as shown in experiments involving depletion of muscle ATP. Also, the maximal capacity of the lysosomal process increased 60-100%, but no changes occurred in the Ca(2+)-dependent or the residual energy-independent proteolytic processes. In muscles from fasted normal and adrenalectomized (ADX) rats, the protein breakdown sensitive to inhibitors of the lysosomal or Ca(2+)-dependent pathways did not differ. However, the ATP-dependent process was 30% slower in muscles from fasted ADX rats. Administering dexamethasone to these animals or incubating their muscles with dexamethasone reversed this defect. During fasting, when the ATP-dependent process rises, muscles show a two- to threefold increase in levels of ubiquitin (Ub) mRNA. However, muscles of ADX animals failed to show this response. Injecting dexamethasone into the fasted ADX animals increased muscle Ub mRNA within 6 h. Thus glucocorticoids activate the ATP-Ub-dependent proteolytic pathway in fasting apparently by enhancing the expression of components of this system such as Ub.

Coordinate Activation of Redox-Dependent ASK1/TGF-β Signaling by a Multiprotein Complex (MPK38, ASK1, SMADs, ZPR9, and TRX) Improves Glucose and Lipid Metabolism in Mice.

PubMed

Seong, Hyun-A; Manoharan, Ravi; Ha, Hyunjung

2016-03-10

To explore the molecular connections between redox-dependent apoptosis signal-regulating kinase 1 (ASK1) and transforming growth factor-β (TGF-β) signaling pathways and to examine the physiological processes in which coordinated regulation of these two signaling pathways plays a critical role. We provide evidence that the ASK1 and TGF-β signaling pathways are interconnected by a multiprotein complex harboring murine protein serine-threonine kinase 38 (MPK38), ASK1, Sma- and Mad-related proteins (SMADs), zinc-finger-like protein 9 (ZPR9), and thioredoxin (TRX) and demonstrate that the activation of either ASK1 or TGF-β activity is sufficient to activate both the redox-dependent ASK1 and TGF-β signaling pathways. Physiologically, the restoration of the downregulated activation levels of ASK1 and TGF-β signaling in genetically and diet-induced obese mice by adenoviral delivery of SMAD3 or ZPR9 results in the amelioration of adiposity, hyperglycemia, hyperlipidemia, and impaired ketogenesis. Our data suggest that the multiprotein complex linking ASK1 and TGF-β signaling pathways may be a potential target for redox-mediated metabolic complications.

Metabolic Respiration Induces AMPK- and Ire1p-Dependent Activation of the p38-Type HOG MAPK Pathway

PubMed Central

Adhikari, Hema; Cullen, Paul J.

2014-01-01

Evolutionarily conserved mitogen activated protein kinase (MAPK) pathways regulate the response to stress as well as cell differentiation. In Saccharomyces cerevisiae, growth in non-preferred carbon sources (like galactose) induces differentiation to the filamentous cell type through an extracellular-signal regulated kinase (ERK)-type MAPK pathway. The filamentous growth MAPK pathway shares components with a p38-type High Osmolarity Glycerol response (HOG) pathway, which regulates the response to changes in osmolarity. To determine the extent of functional overlap between the MAPK pathways, comparative RNA sequencing was performed, which uncovered an unexpected role for the HOG pathway in regulating the response to growth in galactose. The HOG pathway was induced during growth in galactose, which required the nutrient regulatory AMP-dependent protein kinase (AMPK) Snf1p, an intact respiratory chain, and a functional tricarboxylic acid (TCA) cycle. The unfolded protein response (UPR) kinase Ire1p was also required for HOG pathway activation in this context. Thus, the filamentous growth and HOG pathways are both active during growth in galactose. The two pathways redundantly promoted growth in galactose, but paradoxically, they also inhibited each other's activities. Such cross-modulation was critical to optimize the differentiation response. The human fungal pathogen Candida albicans showed a similar regulatory circuit. Thus, an evolutionarily conserved regulatory axis links metabolic respiration and AMPK to Ire1p, which regulates a differentiation response involving the modulated activity of ERK and p38 MAPK pathways. PMID:25356552

Cooperation between STAT5 and phosphatidylinositol 3-kinase in the IL-3-dependent survival of a bone marrow derived cell line.

PubMed

Rosa Santos, S C; Dumon, S; Mayeux, P; Gisselbrecht, S; Gouilleux, F

2000-02-24

Cytokine-dependent activation of distinct signaling pathways is a common scheme thought to be required for the subsequent programmation into cell proliferation and survival. The PI 3-kinase/Akt, Ras/MAP kinase, Ras/NFIL3 and JAK/STAT pathways have been shown to participate in cytokine mediated suppression of apoptosis in various cell types. However the relative importance of these signaling pathways seems to depend on the cellular context. In several cases, individual inhibition of each pathway is not sufficient to completely abrogate cytokine mediated cell survival suggesting that cooperation between these pathways is required. Here we showed that individual inhibition of STAT5, PI 3-kinase or MEK activities did not or weakly affected the IL-3 dependent survival of the bone marrow derived Ba/F3 cell line. However, the simultaneous inhibition of STAT5 and PI 3-kinase activities but not that of STAT5 and MEK reduced the IL-3 dependent survival of Ba/F3. Analysis of the expression of the Bcl-2 members indicated that phosphorylation of Bad and Bcl-x expression which are respectively regulated by the PI 3-kinase/Akt pathway and STAT5 probably explain this cooperation. Furthermore, we showed by co-immunoprecipitation studies and pull down experiments with fusion proteins encoding the GST-SH2 domains of p85 that STAT5 in its phosphorylated form interacts with the p85 subunit of the PI 3-kinase. These results indicate that the activations of STAT5 and the PI 3-kinase by IL-3 in Ba/F3 cells are tightly connected and cooperate to mediate IL-3-dependent suppression of apoptosis by modulating Bad phosphorylation and Bcl-x expression.

E1A enhances cellular sensitivity to DNA-damage-induced apoptosis through PIDD-dependent caspase-2 activation.

PubMed

Radke, Jay R; Siddiqui, Zeba K; Figueroa, Iris; Cook, James L

Expression of the adenoviral protein, E1A, sensitizes mammalian cells to a wide variety of apoptosis-inducing agents through multiple cellular pathways. For example, E1A sensitizes cells to apoptosis induced by TNF-superfamily members by inhibiting NF-kappa B (NF- κ B)-dependent gene expression. In contrast, E1A sensitization to nitric oxide, an inducer of the intrinsic apoptotic pathway, is not dependent upon repression of NF- κ B-dependent transcription but rather is dependent upon caspase-2 activation. The latter observation suggested that E1A-induced enhancement of caspase-2 activation might be a critical factor in cellular sensitization to other intrinsic apoptosis pathway-inducing agents. Etoposide and gemcitabine are two DNA damaging agents that induce intrinsic apoptosis. Here we report that E1A-induced sensitization to both of these agents, like NO, is independent of NF- κ B activation but dependent on caspase-2 activation. The results show that caspase-2 is a key mitochondrial-injuring caspase during etoposide and gemcitabine-induced apoptosis of E1A-positive cells, and that caspase-2 is required for induction of caspase-3 activity by both chemotherapeutic agents. Expression of PIDD was required for caspase-2 activation, mitochondrial injury and enhanced apoptotic cell death. Furthermore, E1A-enhanced sensitivity to injury-induced apoptosis required PIDD cleavage to PIDD-CC. These results define the PIDD/caspase-2 pathway as a key apical, mitochondrial-injuring mechanism in E1A-induced sensitivity of mammalian cells to chemotherapeutic agents.

The contribution of c-Jun N-terminal kinase activation and subsequent Bcl-2 phosphorylation to apoptosis induction in human B-cells is dependent on the mode of action of specific stresses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Muscarella, Donna E.; Bloom, Stephen E.

2008-04-01

The c-Jun N-terminal kinase (JNK) pathway can play paradoxical roles as either a pro-survival or a pro-cell death pathway depending on type of stress and cell type. The goal of the present study was to determine the role of JNK pathway signaling for regulating B-cell apoptosis in two important but contrasting situations-global proteotoxic damage, induced by arsenite and hyperthermia, versus specific microtubule inhibition, induced by the anti-cancer drug vincristine, using the EW36 B-cell line. This cell line over-expresses the Bcl-2 protein and is a useful model to identify treatments that can overcome multi-drug resistance in lymphoid cells. Exposure of EW36more » B-cells to arsenite or lethal hyperthermia resulted in activation of the JNK pathway and induction of apoptosis. However, pharmacological inhibition of the JNK pathway did not inhibit apoptosis, indicating that JNK pathway activation is not required for apoptosis induction by these treatments. In contrast, vincristine treatment of EW36 B-cells resulted in JNK activation and apoptosis that was suppressed by JNK inhibition. A critical difference between the two types of stress treatments was that only vincristine-induced JNK activation resulted in phosphorylation of Bcl-2 at threonine-56, a modification that can block its anti-apoptotic function. Importantly, Bcl-2 phosphorylation was attenuated by JNK inhibition implicating JNK as the upstream kinase. Furthermore, arsenite and hyperthermia treatments activated a p53/p21 pathway associated with apoptosis induction, whereas vincristine did not activate this pathway. These results reveal two stress-activated pathways, one JNK-dependent and another JNK-independent, either of which can bypass Bcl-2 mediated resistance, resulting in cell death.« less

The Diurnal Oscillation of MAP Kinase and Adenylyl Cyclase Activities in the Hippocampus Depends on the SCN

PubMed Central

Phan, Trongha; Chan, Guy; Sindreu, Carlos; Eckel-Mahan, Kristin; Storm, Daniel R.

2011-01-01

Consolidation of hippocampus dependent memory is dependent on activation of the cAMP/ Erk/MAPK signal transduction pathway in the hippocampus. Recently, we discovered that adenylyl cyclase and MAPK activities undergo a circadian oscillation in the hippocampus and that inhibition of this oscillation impairs contextual memory. This suggests the interesting possibility that the persistence of hippocampus-dependent memory depends upon the reactivation of MAPK in the hippocampus during the circadian cycle. A key unanswered question is whether the circadian oscillation of this signaling pathway is intrinsic to the hippocampus or is driven by the master circadian clock in the suprachiasmatic nucleus (SCN). To address this question, we ablated the SCN of mice by electrolytic lesion and examined hippocampus-dependent memory as well as adenylyl cyclase and MAPK activities. Electrolytic lesion of the SCN two days after training for contextual fear memory reduced contextual memory measured two weeks after training indicating that maintenance of contextual memory depends on the SCN. Spatial memory was also compromised in SCN-lesioned mice. Furthermore, the diurnal oscillation of adenylyl cyclase and MAPK activities in the hippocampus was destroyed by lesioning of the SCN. These data suggest that hippocampus-dependent long-term memory is dependent on the SCN-controlled oscillation of the adenylyl cyclase/MAPK pathway in the hippocampus. PMID:21775607

p53-dependent control of cell death by nicastrin: lack of requirement for presenilin-dependent gamma-secretase complex.

PubMed

Pardossi-Piquard, Raphaëlle; Dunys, Julie; Giaime, Emilie; Guillot-Sestier, Marie-Victoire; St George-Hyslop, Peter; Checler, Frédéric; Alves da Costa, Cristine

2009-04-01

Nicastrin (NCT) is a component of the presenilin (PS)-dependent gamma-secretase complexes that liberate amyloid beta-peptides from the beta-Amyloid Precursor Protein. Several lines of evidence indicate that the members of these complexes could also contribute to the control of cell death. Here we show that over-expression of NCT increases the viability of human embryonic kidney (HEK293) cells and decreases staurosporine (STS)- and thapsigargin (TPS)-induced caspase-3 activation in various cell lines from human and neuronal origins by Akt-dependent pathway. NCT lowers p53 expression, transcriptional activity and promoter transactivation and reduces p53 phosphorylation. NCT-associated protection against STS-stimulated cell death was completely abolished by p53 deficiency. Conversely, the depletion of NCT drastically enhances STS-induced caspase-3 activation and p53 pathway and favored p53 nuclear translocation. We examined whether NCT protective function depends on PS-dependent gamma-secretase activity. First, a 29-amino acid deletion known to reduce NCT-dependent amyloid beta-peptide production did not affect NCT-associated protective phenotype. Second, NCT still reduces STS-induced caspase-3 activation in fibroblasts lacking PS1 and PS2. Third, the gamma-secretase inhibitor DFK167 did not affect NCT-mediated reduction of p53 activity. Altogether, our study indicates that NCT controls cell death via phosphoinositide 3-kinase/Akt and p53-dependent pathways and that this function remains independent of the activity and molecular integrity of the gamma-secretase complexes.

Involvement of caspase-dependent and -independent apoptotic pathways in cisplatin-induced apoptosis

NASA Astrophysics Data System (ADS)

Liu, Lei; Zhang, Yingjie; Wang, Xianwang

2009-02-01

Cisplatin, an efficient anticancer agent, can trigger multiple apoptotic pathways in cancer cells. However, the signal transduction pathways in response to cisplatin-based chemotherapy are complicated, and the mechanism is not fully understood. In current study, we showed that, during cisplatin-induced apoptosis of human lung adenocarcinoma cells, both the caspase-dependent and -independent pathways were activated. Herein, we reported that after cisplatin treatment, the activities of caspase-9/-3 were sharply increased; pre-treatment with Z-LEHD-fmk (inhibitor of caspase-9), Z-DEVD-fmk (inhibitor of caspase-3), and Z-VAD-fmk (a pan-caspase inhibitor) increased cell viability and decreased apoptosis, suggesting that caspase-mediated apoptotic pathway was activated following cisplatin treatment. Confocal imaging of the cells transfected with AIF-GFP demonstrated that AIF release occurred about 9 h after cisplatin treatment. The event proceeded progressively over time, coinciding with a nuclear translocation and lasting for more than 2 hours. Down-regulation of AIF by siRNA also significantly increased cell viability and decreased apoptosis, these results suggested that AIF-mediated caspase-independent apoptotic pathway was involved in cispatin-induced apoptosis. In conclusion, the current study demonstrated that both caspase-dependent and -independent apoptotic pathways were involved in cisplatin-induced apoptosis in human lung adenocarcinoma cells.

Murine natural killer immunoreceptors use distinct proximal signaling complexes to direct cell function

PubMed Central

May, Rebecca M.; Okumura, Mariko; Hsu, Chin-Jung; Bassiri, Hamid; Yang, Enjun; Rak, Gregory; Mace, Emily M.; Philip, Naomi H.; Zhang, Weiguo; Baumgart, Tobias; Orange, Jordan S.; Nichols, Kim E.

2013-01-01

Signaling pathways leading to natural killer (NK)–cell effector function are complex and incompletely understood. Here, we investigated the proximal signaling pathways downstream of the immunotyrosine-based activation motif (ITAM) bearing activating receptors. We found that the adaptor molecule SH2 domain-containing leukocyte protein of 76 kD (SLP-76) is recruited to microclusters at the plasma membrane in activated NK cells and that this is required for initiation of downstream signaling and multiple NK-cell effector functions in vitro and in vivo. Surprisingly, we found that 2 types of proximal signaling complexes involving SLP-76 were formed. In addition to the canonical membrane complex formed between SLP-76 and linker for activation of T cells (LAT) family members, a novel LAT family–independent SLP-76–dependent signaling pathway was identified. The LAT family–independent pathway involved the SH2 domain of SLP-76 and adhesion and degranulation-promoting adaptor protein (ADAP). Both the LAT family–dependent and ADAP-dependent pathway contributed to interferon-gamma production and cytotoxicity; however, they were not essential for other SLP-76–dependent events, including phosphorylation of AKT and extracellular signal–related kinase and cellular proliferation. These results demonstrate that NK cells possess an unexpected bifurcation of proximal ITAM-mediated signaling, each involving SLP-76 and contributing to optimal NK-cell function. PMID:23407547

4SC-202 activates ASK1-dependent mitochondrial apoptosis pathway to inhibit hepatocellular carcinoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fu, Meili, E-mail: fumeilidrlinyi@tom.com; Wan, Fuqiang; Li, Zhengling

The aim of the present study is to investigate the potential anti-hepatocellular carcinoma (HCC) cell activity by 4SC-202, a novel class I HDAC inhibitor (HDACi). The associated signaling mechanisms were also analyzed. We showed that 4SC-202 treatment induced potent cytotoxic and proliferation–inhibitory activities against established HCC cell lines (HepG2, HepB3, SMMC-7721) and patient-derived primary HCC cells. Further, adding 4SC-202 in HCC cells activated mitochondrial apoptosis pathway, which was evidenced by mitochondrial permeability transition pore (mPTP) opening, cytochrome C cytosol release and caspase-3/-9 activation. Inhibition of this apoptosis pathway, by caspase-3/-9 inhibitors, mPTP blockers, or by shRNA-mediated knockdown of cyclophilin-D (Cyp-D,more » a key component of mPTP), significantly attenuated 4SC-202-induced HCC cell death and apoptosis. Reversely, over-expression of Cyp-D enhanced 4SC-202's sensitivity in HCC cells. Further studies showed that 4SC-202 induced apoptosis signal-regulating kinase 1 (ASK1) activation, causing it translocation to mitochondria and physical association with Cyp-D. This mitochondrial ASK1-Cyp-D complexation appeared required for mediating 4SC-202-induced apoptosis activation. ASK1 stable knockdown by targeted-shRNAs largely inhibited 4SC-202-induced mPTP opening, cytochrome C release, and following HCC cell apoptotic death. Together, we suggest that 4SC-202 activates ASK1-dependent mitochondrial apoptosis pathway to potently inhibit human HCC cells. - Highlights: • 4SC-202 exerts potent anti-proliferative and cytotoxic activity against established/primary HCC cells. • SC-202-induced anti-HCC cell activity relies on caspase-dependent apoptosis activation. • 4SC-202 activates Cyp-D-dependent mitochondrial apoptosis pathway in HCC cells. • 4SC-202 activates ASK1 in HCC cells, causing it translocation to mitochondria. • Mitochondrial ASK1-Cyp-D complexation mediates 4SC-202's activity in HCC cells.« less

Selectins and chemokines use shared and distinct signals to activate β2 integrins in neutrophils

PubMed Central

Yago, Tadayuki; Zhang, Nan; Zhao, Liang; Abrams, Charles S.

2018-01-01

Rolling neutrophils receive signals while engaging P- and E-selectin and chemokines on inflamed endothelium. Selectin signaling activates β2 integrins to slow rolling velocities. Chemokine signaling activates β2 integrins to cause arrest. Despite extensive study, key aspects of these signaling cascades remain unresolved. Using complementary in vitro and in vivo assays, we found that selectin and chemokine signals in neutrophils triggered Rap1a-dependent and phosphatidylinositol-4-phosphate 5-kinase γ (PIP5Kγ90)–dependent pathways that induce integrin-dependent slow rolling and arrest. Interruption of both pathways, but not either pathway alone, blocked talin-1 recruitment to and activation of integrins. An isoform of PIP5Kγ90 lacking the talin-binding domain (PIP5Kγ87) could not activate integrins. Chemokines, but not selectins, used phosphatidylinositol-4,5-bisphosphate 3-kinase γ (PI3Kγ) in cooperation with Rap1a to mediate integrin-dependent slow rolling (at low chemokine concentrations), as well as arrest (at high chemokine concentrations). High levels of chemokines activated β2 integrins without selectin signals. When chemokines were limiting, they synergized with selectins to activate β2 integrins. PMID:29592875

Selectins and chemokines use shared and distinct signals to activate β2 integrins in neutrophils.

PubMed

Yago, Tadayuki; Zhang, Nan; Zhao, Liang; Abrams, Charles S; McEver, Rodger P

2018-04-10

Rolling neutrophils receive signals while engaging P- and E-selectin and chemokines on inflamed endothelium. Selectin signaling activates β2 integrins to slow rolling velocities. Chemokine signaling activates β2 integrins to cause arrest. Despite extensive study, key aspects of these signaling cascades remain unresolved. Using complementary in vitro and in vivo assays, we found that selectin and chemokine signals in neutrophils triggered Rap1a-dependent and phosphatidylinositol-4-phosphate 5-kinase γ (PIP5Kγ90)-dependent pathways that induce integrin-dependent slow rolling and arrest. Interruption of both pathways, but not either pathway alone, blocked talin-1 recruitment to and activation of integrins. An isoform of PIP5Kγ90 lacking the talin-binding domain (PIP5Kγ87) could not activate integrins. Chemokines, but not selectins, used phosphatidylinositol-4,5-bisphosphate 3-kinase γ (PI3Kγ) in cooperation with Rap1a to mediate integrin-dependent slow rolling (at low chemokine concentrations), as well as arrest (at high chemokine concentrations). High levels of chemokines activated β2 integrins without selectin signals. When chemokines were limiting, they synergized with selectins to activate β2 integrins. © 2018 by The American Society of Hematology.

Protein Kinase Cα Modulates Estrogen-Receptor-Dependent Transcription and Proliferation in Endometrial Cancer Cells

PubMed Central

Thorne, Alicia M.; Jackson, Twila A.; Willis, Van C.; Bradford, Andrew P.

2013-01-01

Endometrial cancer is the most common invasive gynecologic malignancy in developed countries. The most prevalent endometrioid tumors are linked to excessive estrogen exposure and hyperplasia. However, molecular mechanisms and signaling pathways underlying their etiology and pathophysiology remain poorly understood. We have shown that protein kinase Cα (PKCα) is aberrantly expressed in endometrioid tumors and is an important mediator of endometrial cancer cell survival, proliferation, and invasion. In this study, we demonstrate that expression of active, myristoylated PKCα conferred ligand-independent activation of estrogen-receptor- (ER-) dependent promoters and enhanced responses to estrogen. Conversely, knockdown of PKCα reduced ER-dependent gene expression and inhibited estrogen-induced proliferation of endometrial cancer cells. The ability of PKCα to potentiate estrogen activation of ER-dependent transcription was attenuated by inhibitors of phosphoinositide 3-kinase (PI3K) and Akt. Evidence suggests that PKCα and estrogen signal transduction pathways functionally interact, to modulate ER-dependent growth and transcription. Thus, PKCα signaling, via PI3K/Akt, may be a critical element of the hyperestrogenic environment and activation of ER that is thought to underlie the development of estrogen-dependent endometrial hyperplasia and malignancy. PKCα-dependent pathways may provide much needed prognostic markers of aggressive disease and novel therapeutic targets in ER positive tumors. PMID:23843797

BECN1-dependent CASP2 incomplete autophagy induction by binding to rabies virus phosphoprotein.

PubMed

Liu, Juan; Wang, Hailong; Gu, Jinyan; Deng, Tingjuan; Yuan, Zhuangchuan; Hu, Boli; Xu, Yunbin; Yan, Yan; Zan, Jie; Liao, Min; DiCaprio, Erin; Li, Jianrong; Su, Shuo; Zhou, Jiyong

2017-04-03

Autophagy is an essential component of host immunity and used by viruses for survival. However, the autophagy signaling pathways involved in virus replication are poorly documented. Here, we observed that rabies virus (RABV) infection triggered intracellular autophagosome accumulation and results in incomplete autophagy by inhibiting autophagy flux. Subsequently, we found that RABV infection induced the reduction of CASP2/caspase 2 and the activation of AMP-activated protein kinase (AMPK)-AKT-MTOR (mechanistic target of rapamycin) and AMPK-MAPK (mitogen-activated protein kinase) pathways. Further investigation revealed that BECN1/Beclin 1 binding to viral phosphoprotein (P) induced an incomplete autophagy via activating the pathways CASP2-AMPK-AKT-MTOR and CASP2-AMPK-MAPK by decreasing CASP2. Taken together, our data first reveals a crosstalk of BECN1 and CASP2-dependent autophagy pathways by RABV infection.

Cardiotoxic and Cardioprotective Features of Chronic β-adrenergic Signaling

PubMed Central

Zhang, Xiaoying; Szeto, Christopher; Gao, Erhe; Tang, Mingxin; Jin, Jianguo; Fu, Qin; Makarewich, Catherine; Ai, Xiaojie; Li, Ying; Tang, Allen; Wang, Jenny; Gao, Hui; Wang, Fang; Ge, Xinyi Joy; Kunapuli, Satya P.; Zhou, Lin; Zeng, Chunyu; Xiang, Kevin Yang; Chen, Xiongwen

2012-01-01

Rationale In the failing heart, persistent β-adrenergic receptor (βAR) activation is thought to induce myocyte death by protein kinase A (PKA)-dependent and PKA-independent activation of calcium/calmodulin-dependent kinase II (CaMKII). β-Adrenergic signaling pathways are also capable of activating cardioprotective mechanisms. Objective This study used a novel PKA inhibitor peptide (PKI) to inhibit PKA activity to test the hypothesis that βAR signaling causes cell death through PKA-dependent pathways and cardioprotection through PKA-independent pathways. Methods and Results In PKI transgenic mice, chronic isoproterenol (ISO) failed to induce cardiac hypertrophy, fibrosis, myocyte apoptosis and depressed cardiac function. In cultured adult feline ventricular myocytes (AFVMs), PKA inhibition protected myocytes from death induced by β1-AR agonists by preventing cytosolic and SR Ca2+ overload and CaMKII activation. PKA inhibition revealed a cardioprotective role of β-adrenergic signaling via cAMP/EPAC /Rap1/Rac/ERK pathway. Selective PKA inhibition causes protection in the heart after myocardial infarction (MI) that was superior to β-blocker therapy. Conclusion These results suggest that selective block of PKA could be a novel heart failure therapy. PMID:23104882

Snf1 Phosphorylates Adenylate Cyclase and Negatively Regulates Protein Kinase A-dependent Transcription in Saccharomyces cerevisiae.

PubMed

Nicastro, Raffaele; Tripodi, Farida; Gaggini, Marco; Castoldi, Andrea; Reghellin, Veronica; Nonnis, Simona; Tedeschi, Gabriella; Coccetti, Paola

2015-10-09

In eukaryotes, nutrient availability and metabolism are coordinated by sensing mechanisms and signaling pathways, which influence a broad set of cellular functions such as transcription and metabolic pathways to match environmental conditions. In yeast, PKA is activated in the presence of high glucose concentrations, favoring fast nutrient utilization, shutting down stress responses, and boosting growth. On the contrary, Snf1/AMPK is activated in the presence of low glucose or alternative carbon sources, thus promoting an energy saving program through transcriptional activation and phosphorylation of metabolic enzymes. The PKA and Snf1/AMPK pathways share common downstream targets. Moreover, PKA has been reported to negatively influence the activation of Snf1/AMPK. We report a new cross-talk mechanism with a Snf1-dependent regulation of the PKA pathway. We show that Snf1 and adenylate cyclase (Cyr1) interact in a nutrient-independent manner. Moreover, we identify Cyr1 as a Snf1 substrate and show that Snf1 activation state influences Cyr1 phosphorylation pattern, cAMP intracellular levels, and PKA-dependent transcription. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Chronic administration of aripiprazole activates GSK3β-dependent signalling pathways, and up-regulates GABAA receptor expression and CREB1 activity in rats.

PubMed

Pan, Bo; Huang, Xu-Feng; Deng, Chao

2016-07-20

Aripiprazole is a D2-like receptor (D2R) partial agonist with a favourable clinical profile. Previous investigations indicated that acute and short-term administration of aripiprazole had effects on PKA activity, GSK3β-dependent pathways, GABAA receptors, NMDA receptor and CREB1 in the brain. Since antipsychotics are used chronically in clinics, the present study investigated the long-term effects of chronic oral aripiprazole treatment on these cellular signalling pathways, in comparison with haloperidol (a D2R antagonist) and bifeprunox (a potent D2R partial agonist). We found that the Akt-GSK3β pathway was activated by aripiprazole and bifeprunox in the prefrontal cortex; NMDA NR2A levels were reduced by aripiprazole and haloperidol. In the nucleus accumbens, all three drugs increased Akt-GSK3β signalling; in addition, both aripiprazole and haloperidol, but not bifeprunox, increased the expression of Dvl-3, β-catenin and GABAA receptors, NMDA receptor subunits, as well as CREB1 phosphorylation levels. The results suggest that chronic oral administration of aripiprazole affects schizophrenia-related cellular signalling pathways and markers (including Akt-GSK3β signalling, Dvl-GSK3β-β-catenin signalling, GABAA receptor, NMDA receptor and CREB1) in a brain-region-dependent manner; the selective effects of aripiprazole on these signalling pathways might be associated with its unique clinical effects.

Activation of mPTP-dependent mitochondrial apoptosis pathway by a novel pan HDAC inhibitor resminostat in hepatocellular carcinoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fu, Meili; Shi, Wenhong; Li, Zhengling

Over-expression and aberrant activation of histone deacetylases (HDACs) are often associated with poor prognosis of hepatocellular carcinoma (HCC). Here, we evaluated the potential anti-hepatocellular carcinoma (HCC) cell activity by resminostat, a novel pan HDAC inhibitor (HDACi). We demonstrated that resminostat induced potent cytotoxic and anti-proliferative activity against established HCC cell lines (HepG2, HepB3, SMMC-7721) and patient-derived primary HCC cells. Further, resminostat treatment in HCC cells activated mitochondrial permeability transition pore (mPTP)-dependent apoptosis pathway, which was evidenced by physical association of cyclophilin-D and adenine nucleotide translocator 1 (ANT-1), mitochondrial depolarization, cytochrome C release and caspase-9 activation. Intriguingly, the mPTP blockers (sanglifehrinmore » A and cyclosporine A), shRNA knockdown of cyclophilin-D or the caspase-9 inhibitor dramatically attenuated resminostat-induced HCC cell apoptosis and cytotoxicity. Reversely, HCC cells with exogenous cyclophilin-D over-expression were hyper-sensitive to resminostat. Intriguingly, a low concentration of resminostat remarkably potentiated sorafenib-induced mitochondrial apoptosis pathway activation, leading to a profound cytotoxicity in HCC cells. The results of this preclinical study indicate that resminostat (or plus sorafenib) could be further investigated as a valuable anti-HCC strategy. - Highlights: • Resminostat inhibits human HCC cell survival and proliferation. • Resminostat activates mPTP-dependent mitochondrial apoptosis pathway in HCC cells. • Resminostat potentiates sorafenib-induced mitochondrial apoptosis pathway activation. • mPTP or caspase-9 inhibition attenuates apoptosis by resminostat or plus sorafenib.« less

Phagocytosis-dependent activation of a TLR9–BTK–calcineurin–NFAT pathway co-ordinates innate immunity to Aspergillus fumigatus

PubMed Central

Herbst, Susanne; Shah, Anand; Mazon Moya, Maria; Marzola, Vanessa; Jensen, Barbara; Reed, Anna; Birrell, Mark A; Saijo, Shinobu; Mostowy, Serge; Shaunak, Sunil; Armstrong-James, Darius

2015-01-01

Transplant recipients on calcineurin inhibitors are at high risk of invasive fungal infection. Understanding how calcineurin inhibitors impair fungal immunity is a key priority for defining risk of infection. Here, we show that the calcineurin inhibitor tacrolimus impairs clearance of the major mould pathogen Aspergillus fumigatus from the airway, by inhibiting macrophage inflammatory responses. This leads to defective early neutrophil recruitment and fungal clearance. We confirm these findings in zebrafish, showing an evolutionarily conserved role for calcineurin signalling in neutrophil recruitment during inflammation. We find that calcineurin–NFAT activation is phagocytosis dependent and collaborates with NF-κB for TNF-α production. For yeast zymosan particles, activation of macrophage calcineurin–NFAT occurs via the phagocytic Dectin-1–spleen tyrosine kinase pathway, but for A. fumigatus, activation occurs via a phagosomal TLR9-dependent and Bruton's tyrosine kinase-dependent signalling pathway that is independent of MyD88. We confirm the collaboration between NFAT and NF-κB for TNF-α production in primary alveolar macrophages. These observations identify inhibition of a newly discovered macrophage TLR9–BTK–calcineurin–NFAT signalling pathway as a key immune defect that leads to organ transplant-related invasive aspergillosis. PMID:25637383

Src-family-tyrosine kinase Lyn is critical for TLR2-mediated NF-κB activation through the PI 3-kinase signaling pathway.

PubMed

Toubiana, Julie; Rossi, Anne-Lise; Belaidouni, Nadia; Grimaldi, David; Pene, Frederic; Chafey, Philippe; Comba, Béatrice; Camoin, Luc; Bismuth, Georges; Claessens, Yann-Erick; Mira, Jean-Paul; Chiche, Jean-Daniel

2015-10-01

TLR2 has a prominent role in host defense against a wide variety of pathogens. Stimulation of TLR2 triggers MyD88-dependent signaling to induce NF-κB translocation, and activates a Rac1-PI 3-kinase dependent pathway that leads to transactivation of NF-κB through phosphorylation of the P65 NF-κB subunit. This transactivation pathway involves tyrosine phosphorylations. The role of the tyrosine kinases in TLR signaling is controversial, with discrepancies between studies using only chemical inhibitors and knockout mice. Here, we show the involvement of the tyrosine-kinase Lyn in TLR2-dependent activation of NF-κB in human cellular models, by using complementary inhibition strategies. Stimulation of TLR2 induces the formation of an activation cluster involving TLR2, CD14, PI 3-kinase and Lyn, and leads to the activation of AKT. Lyn-dependent phosphorylation of the p110 catalytic subunit of PI 3-kinase is essential to the control of PI 3-kinase biological activity upstream of AKT and thereby to the transactivation of NF-κB. Thus, Lyn kinase activity is crucial in TLR2-mediated activation of the innate immune response in human mononuclear cells. © The Author(s) 2015.

Ubiquitin-Dependent Regulation of the Mammalian Hippo Pathway: Therapeutic Implications for Cancer.

PubMed

Nguyen, Thanh Hung; Kugler, Jan-Michael

2018-04-17

The Hippo pathway serves as a key barrier for oncogenic transformation. It acts by limiting the activity of the proto-oncogenes YAP and TAZ. Reduced Hippo signaling and elevated YAP/TAZ activities are frequently observed in various types of tumors. Emerging evidence suggests that the ubiquitin system plays an important role in regulating Hippo pathway activity. Deregulation of ubiquitin ligases and of deubiquitinating enzymes has been implicated in increased YAP/TAZ activity in cancer. In this article, we review recent insights into the ubiquitin-mediated regulation of the mammalian Hippo pathway, its deregulation in cancer, and possibilities for targeting the Hippo pathway through the ubiquitin system.

Role of the ceramide-signaling pathways in ionizing radiation-induced apoptosis.

PubMed

Vit, Jean-Philippe; Rosselli, Filippo

2003-11-27

Ionizing radiations (IR) exposure leads to damage on several cellular targets. How signals from different targets are integrated to determine the cell fate remains a controversial issue. Understanding the pathway(s) responsible(s) for the cell killing effect of the IR exposure is of prime importance in light of using radiations as anticancer agent or as diagnostic tool. In this study, we have established that IR-induced cell damage initiates two independent signaling pathways that lead to a biphasic intracellular ceramide increase. A transitory increase of ceramide is observed within minutes after IR exposure as a consequence of DNA damage-independent acid sphingomyelinase activation. Several hours after irradiation, a second wave of ceramide accumulation is observed depending on the DNA damage-dependent activation of ceramide synthase, which requires a signaling pathway involving ATM. Importantly, we have demonstrated that the late ceramide accumulation is also dependent on the first one and is rate limiting for the apoptotic process induced by IR. In conclusion, our observations suggest that ceramide is a major determinant of the IR-induced apoptotic process at the cross-point of different signal transduction pathways.

The IgE-dependent pathway in allergic transfusion reactions: involvement of donor blood allergens other than plasma proteins.

PubMed

Matsuyama, Nobuki; Yasui, Kazuta; Amakishi, Etsuko; Hayashi, Tomoya; Kuroishi, Ayumu; Ishii, Hiroyuki; Matsukura, Harumichi; Tani, Yoshihiko; Furuta, Rika A; Hirayama, Fumiya

2015-07-01

On transfusion, several plasma proteins can cause anaphylaxis in patients deficient in the corresponding plasma proteins. However, little is known about other allergens, which are encountered much more infrequently. Although it has been speculated that an allergen-independent pathway underlying allergic transfusion reactions (ATRs) is elicited by biological response modifiers accumulated in blood components during storage, the exact mechanisms remain unresolved. Furthermore, it is difficult even to determine whether ATRs are induced via allergen-dependent or allergen-independent pathways. To distinguish these two pathways in ATR cases, we established a basophil activation test, in which the basophil-activating ability of supernatants of residual transfused blood of ATR cases to whole blood basophils was assessed in the presence or absence of dasatinib, an inhibitor of IgE-mediated basophil activation. Three of 37 supernatants from the platelet concentrates with ATRs activated panel blood basophils in the absence, but not in the presence, of dasatinib. The basophil activation was inhibited by treatment of anti-fish collagen I MoAb in one case, suggesting that the involvement of fish allergens may have been present in donor plasma. We concluded that unknown non-plasma proteins, some of which had epitopes similar to fish antigens, in blood component may be involved in ATRs via an allergen/IgE-dependent pathway.

A PKA survival pathway inhibited by DPT-PKI, a new specific cell permeable PKA inhibitor, is induced by T. annulata in parasitized B-lymphocytes.

PubMed

Guergnon, Julien; Dessauge, Frederic; Traincard, François; Cayla, Xavier; Rebollo, Angelita; Bost, Pierre Etienne; Langsley, Gordon; Garcia, Alphonse

2006-08-01

T. annulata, an intracellular pathogenic parasite of the Aplicomplexa protozoan family infects bovine B-lymphocytes and macrophages. Parasitized cells that become transformed survive and proliferate independently of exogenous growth factors. In the present study, we used the isogenic non parasitized BL3 and parasitized TBL3 B cell lines, as a model to evaluate the contribution of two-major PI3-K- and PKA-dependent anti-apoptotic pathways in the survival of T. annulata parasitized B lymphocytes. We found that T. annulata increases PKA activity, induces over-expression of the catalytic subunit and down-regulates the pro-survival phosphorylation state of Akt/PKB. Consistent with a role of PKA activation in survival, two pharmacological inhibitors H89 and KT5720 ablate PKA-dependent survival of parasitized cells. To specifically inhibit PKA pro-survival pathways we linked the DPTsh1 peptide shuttle sequence to PKI(5-24) and we generated DPT-PKI, a cell permeable PKI. DPT-PKI specifically inhibited PKA activity in bovine cell extracts and, as expected, also inhibited the PKA-dependent survival of T. annulata parasitized TBL3 cells. Thus, parasite-dependent constitutive activation of PKA in TBL3 cells generates an anti-apoptotic pathway that can protect T. annulata-infected B cells from apoptosis. These results also indicate that DPT-PKI could be a powerful tool to inhibit PKA pathways in other cell types.

HMMR acts in the PLK1-dependent spindle positioning pathway and supports neural development

PubMed Central

Jiang, Jihong; Kuan, Chia-Wei; Fotovati, Abbas; Chu, Tony LH; He, Zhengcheng; Lengyell, Tess C; Li, Huaibiao; Kroll, Torsten; Li, Amanda M; Goldowitz, Daniel; Frappart, Lucien; Ploubidou, Aspasia; Patel, Millan S; Pilarski, Linda M; Simpson, Elizabeth M; Lange, Philipp F; Allan, Douglas W

2017-01-01

Oriented cell division is one mechanism progenitor cells use during development and to maintain tissue homeostasis. Common to most cell types is the asymmetric establishment and regulation of cortical NuMA-dynein complexes that position the mitotic spindle. Here, we discover that HMMR acts at centrosomes in a PLK1-dependent pathway that locates active Ran and modulates the cortical localization of NuMA-dynein complexes to correct mispositioned spindles. This pathway was discovered through the creation and analysis of Hmmr-knockout mice, which suffer neonatal lethality with defective neural development and pleiotropic phenotypes in multiple tissues. HMMR over-expression in immortalized cancer cells induces phenotypes consistent with an increase in active Ran including defects in spindle orientation. These data identify an essential role for HMMR in the PLK1-dependent regulatory pathway that orients progenitor cell division and supports neural development. PMID:28994651

Spatial separation of two different pathways accounting for the generation of calcium signals in astrocytes.

PubMed

Oschmann, Franziska; Mergenthaler, Konstantin; Jungnickel, Evelyn; Obermayer, Klaus

2017-02-01

Astrocytes integrate and process synaptic information and exhibit calcium (Ca2+) signals in response to incoming information from neighboring synapses. The generation of Ca2+ signals is mostly attributed to Ca2+ release from internal Ca2+ stores evoked by an elevated metabotropic glutamate receptor (mGluR) activity. Different experimental results associated the generation of Ca2+ signals to the activity of the glutamate transporter (GluT). The GluT itself does not influence the intracellular Ca2+ concentration, but it indirectly activates Ca2+ entry over the membrane. A closer look into Ca2+ signaling in different astrocytic compartments revealed a spatial separation of those two pathways. Ca2+ signals in the soma are mainly generated by Ca2+ release from internal Ca2+ stores (mGluR-dependent pathway). In astrocytic compartments close to the synapse most Ca2+ signals are evoked by Ca2+ entry over the plasma membrane (GluT-dependent pathway). This assumption is supported by the finding, that the volume ratio between the internal Ca2+ store and the intracellular space decreases from the soma towards the synapse. We extended a model for mGluR-dependent Ca2+ signals in astrocytes with the GluT-dependent pathway. Additionally, we included the volume ratio between the internal Ca2+ store and the intracellular compartment into the model in order to analyze Ca2+ signals either in the soma or close to the synapse. Our model results confirm the spatial separation of the mGluR- and GluT-dependent pathways along the astrocytic process. The model allows to study the binary Ca2+ response during a block of either of both pathways. Moreover, the model contributes to a better understanding of the impact of channel densities on the interaction of both pathways and on the Ca2+ signal.

Dynamic synchronization of ongoing neuronal activity across spinal segments regulates sensory information flow

PubMed Central

Contreras-Hernández, E; Chávez, D; Rudomin, P

2015-01-01

Previous studies on the correlation between spontaneous cord dorsum potentials recorded in the lumbar spinal segments of anaesthetized cats suggested the operation of a population of dorsal horn neurones that modulates, in a differential manner, transmission along pathways mediating Ib non-reciprocal postsynaptic inhibition and pathways mediating primary afferent depolarization and presynaptic inhibition. In order to gain further insight into the possible neuronal mechanisms that underlie this process, we have measured changes in the correlation between the spontaneous activity of individual dorsal horn neurones and the cord dorsum potentials associated with intermittent activation of these inhibitory pathways. We found that high levels of neuronal synchronization within the dorsal horn are associated with states of incremented activity along the pathways mediating presynaptic inhibition relative to pathways mediating Ib postsynaptic inhibition. It is suggested that ongoing changes in the patterns of functional connectivity within a distributed ensemble of dorsal horn neurones play a relevant role in the state-dependent modulation of impulse transmission along inhibitory pathways, among them those involved in the central control of sensory information. This feature would allow the same neuronal network to be involved in different functional tasks. Key points We have examined, in the spinal cord of the anaesthetized cat, the relationship between ongoing correlated fluctuations of dorsal horn neuronal activity and state-dependent activation of inhibitory reflex pathways. We found that high levels of synchronization between the spontaneous activity of dorsal horn neurones occur in association with the preferential activation of spinal pathways leading to primary afferent depolarization and presynaptic inhibition relative to activation of pathways mediating Ib postsynaptic inhibition. It is suggested that changes in synchronization of ongoing activity within a distributed network of dorsal horn neurones play a relevant role in the configuration of structured (non-random) patterns of functional connectivity that shape the interaction of sensory inputs with spinal reflex pathways subserving different functional tasks. PMID:25653206

Dissecting Bacterial Cell Wall Entry and Signaling in Eukaryotic Cells: an Actin-Dependent Pathway Parallels Platelet-Activating Factor Receptor-Mediated Endocytosis.

PubMed

Loh, Lip Nam; Gao, Geli; Tuomanen, Elaine I

2017-01-03

The Gram-positive bacterial cell wall (CW) peptidoglycan-teichoic acid complex is released into the host environment during bacterial metabolism or death. It is a highly inflammatory Toll-like receptor 2 (TLR2) ligand, and previous in vivo studies have demonstrated its ability to recapitulate pathological features of pneumonia and meningitis. We report that an actin-dependent pathway is involved in the internalization of the CW by epithelial and endothelial cells, in addition to the previously described platelet-activating factor receptor (PAFr)-dependent uptake pathway. Unlike the PAFr-dependent pathway, which is mediated by clathrin and dynamin and does not lead to signaling, the alternative pathway is sensitive to 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and engenders Rac1, Cdc42, and phosphatidylinositol 3-kinase (PI3K) signaling. Upon internalization by this macropinocytosis-like pathway, CW is trafficked to lysosomes. Intracellular CW trafficking is more complex than previously recognized and suggests multiple points of interaction with and without innate immune signaling. Streptococcus pneumoniae is a major human pathogen infecting the respiratory tract and brain. It is an established model organism for understanding how infection injures the host. During infection or bacterial growth, bacteria shed their cell wall (CW) into the host environment and trigger inflammation. A previous study has shown that CW enters and crosses cell barriers by interacting with a receptor on the surfaces of host cells, termed platelet-activating factor receptor (PAFr). In the present study, by using cells that are depleted of PAFr, we identified a second pathway with features of macropinocytosis, which is a receptor-independent fluid uptake mechanism by cells. Each pathway contributes approximately the same amount of cell wall trafficking, but the PAFr pathway is silent, while the new pathway appears to contribute to the host inflammatory response to CW insult. Copyright © 2017 Loh et al.

Ligand-independent pathway that controls stability of interferon alpha receptor

PubMed Central

Liu, Jianghuai; Plotnikov, Alexander; Banerjee, Anamika; Kumar, K.G. Suresh; Ragimbeau, Josiane; Marijanovic, Zrinka; Baker, Darren P.; Pellegrini, Sandra; Fuchs, Serge Y.

2008-01-01

SUMMARY Ligand-specific negative regulation of cytokine-induced signaling relies on down regulation of the cytokine receptors. Down regulation of the IFNAR1 sub-unit of the Type I interferon (IFN) receptor proceeds via lysosomal receptor proteolysis, which is triggered by ubiquitination that depends on IFNAR1 serine phosphorylation. While IFN-inducible phosphorylation, ubiquitination and degradation requires the catalytic activity of the Tyk2 Janus kinase, here we found the ligand- and Tyk2-independent pathway that promotes IFNAR1 phosphorylation, ubiquitination, and degradation when IFNAR1 is expressed at high levels. A major cellular kinase activity that is responsible for IFNAR1 phosphorylation in vitro does not depend on either ligand or Tyk2 activity. Inhibition of ligand-independent IFNAR1 degradation suppresses cell proliferation. We discuss the signaling events that might lead to ubiquitination and degradation of IFNAR1 via ligand-dependent and independent pathways and their potential physiologic significance. PMID:18166147

Induction of Cardiac Fibrosis by β-Blocker in G Protein-independent and G Protein-coupled Receptor Kinase 5/β-Arrestin2-dependent Signaling Pathways*

PubMed Central

Nakaya, Michio; Chikura, Satsuki; Watari, Kenji; Mizuno, Natsumi; Mochinaga, Koji; Mangmool, Supachoke; Koyanagi, Satoru; Ohdo, Shigehiro; Sato, Yoji; Ide, Tomomi; Nishida, Motohiro; Kurose, Hitoshi

2012-01-01

G-protein coupled receptors (GPCRs) have long been known as receptors that activate G protein-dependent cellular signaling pathways. In addition to the G protein-dependent pathways, recent reports have revealed that several ligands called “biased ligands” elicit G protein-independent and β-arrestin-dependent signaling through GPCRs (biased agonism). Several β-blockers are known as biased ligands. All β-blockers inhibit the binding of agonists to the β-adrenergic receptors. In addition to β-blocking action, some β-blockers are reported to induce cellular responses through G protein-independent and β-arrestin-dependent signaling pathways. However, the physiological significance induced by the β-arrestin-dependent pathway remains much to be clarified in vivo. Here, we demonstrate that metoprolol, a β1-adrenergic receptor-selective blocker, could induce cardiac fibrosis through a G protein-independent and β-arrestin2-dependent pathway. Metoprolol, a β-blocker, increased the expression of fibrotic genes responsible for cardiac fibrosis in cardiomyocytes. Furthermore, metoprolol induced the interaction between β1-adrenergic receptor and β-arrestin2, but not β-arrestin1. The interaction between β1-adrenergic receptor and β-arrestin2 by metoprolol was impaired in the G protein-coupled receptor kinase 5 (GRK5)-knockdown cells. Metoprolol-induced cardiac fibrosis led to cardiac dysfunction. However, the metoprolol-induced fibrosis and cardiac dysfunction were not evoked in β-arrestin2- or GRK5-knock-out mice. Thus, metoprolol is a biased ligand that selectively activates a G protein-independent and GRK5/β-arrestin2-dependent pathway, and induces cardiac fibrosis. This study demonstrates the physiological importance of biased agonism, and suggests that G protein-independent and β-arrestin-dependent signaling is a reason for the diversity of the effectiveness of β-blockers. PMID:22888001

Use Dependence of Heat Sensitivity of Vanilloid Receptor TRPV2

PubMed Central

Liu, Beiying; Qin, Feng

2016-01-01

Thermal TRP channels mediate temperature transduction and pain sensation. The vanilloid receptor TRPV2 is involved in detection of noxious heat in a subpopulation of high-threshold nociceptors. It also plays a critical role in development of thermal hyperalgesia, but the underlying mechanism remains uncertain. Here we analyze the heat sensitivity of the TRPV2 channel. Heat activation of the channel exhibits strong use dependence. Prior heat activation can profoundly alter its subsequent temperature responsiveness, causing decreases in both temperature activation threshold and slope sensitivity of temperature dependence while accelerating activation time courses. Notably, heat and agonist activations differ in cross use-dependence. Prior heat stimulation can dramatically sensitize agonist responses, but not conversely. Quantitative analyses indicate that the use dependence in heat sensitivity is pertinent to the process of temperature sensing by the channel. The use dependence of TRPV2 reveals that the channel can have a dynamic temperature sensitivity. The temperature sensing structures within the channel have multiple conformations and the temperature activation pathway is separate from the agonist activation pathway. Physiologically, the use dependence of TRPV2 confers nociceptors with a hypersensitivity to heat and thus provides a mechanism for peripheral thermal hyperalgesia. PMID:27074678

Dual targeting of HER3 and MEK may overcome HER3-dependent drug-resistance of colon cancers

PubMed Central

Bon, Giulia; Loria, Rossella; Amoreo, Carla Azzurra; Verdina, Alessandra; Sperduti, Isabella; Mastrofrancesco, Arianna; Soddu, Silvia; Diodoro, Maria Grazia; Mottolese, Marcella; Todaro, Matilde; Stassi, Giorgio; Milella, Michele; De Maria, Ruggero; Falcioni, Rita

2017-01-01

Although the medical treatment of colorectal cancer has evolved greatly in the last years, a significant portion of early-stage patients develops recurrence after therapies. The current clinical trials are directed to evaluate new drug combinations and treatment schedules. By the use of patient-derived or established colon cancer cell lines, we found that the tyrosine kinase receptor HER3 is involved in the mechanisms of resistance to therapies. In agreement, the immunohistochemical analysis of total and phospho-HER3 expression in 185 colorectal cancer specimens revealed a significant correlation with lower disease-free survival. Targeting HER3 by the use of the monoclonal antibody patritumab we found induction of growth arrest in all cell lines. Despite the high efficiency of patritumab in abrogating the HER3-dependent activation of PI3K pathway, the HER2 and EGFR-dependent MAPK pathway is activated as a compensatory mechanism. Interestingly, we found that the MEK-inhibitor trametinib inhibits, as expected, the MAPK pathway but induces the HER3-dependent activation of PI3K pathway. The combined treatment results in the abrogation of both PI3K and MAPK pathways and in a significant reduction of cell proliferation and survival. These data suggest a new strategy of therapy for HER3-overexpressing colon cancers. PMID:29312543

Insulin-like growth factor-1 suppresses the Myostatin signaling pathway during myogenic differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Retamales, A.; Zuloaga, R.; Valenzuela, C.A.

Myogenic differentiation is a complex and well-coordinated process for generating mature skeletal muscle fibers. This event is autocrine/paracrine regulated by growth factors, principally Myostatin (MSTN) and Insulin-like Growth Factor-1 (IGF-1). Myostatin, a member of the transforming growth factor-β superfamily, is a negative regulator of skeletal muscle growth in vertebrates that exerts its inhibitory function by activating Smad transcription factors. In contrast, IGF-1 promotes the differentiation of skeletal myoblasts by activating the PI3K/Akt signaling pathway. This study reports on a novel functional crosstalk between the IGF-1 and MSTN signaling pathways, as mediated through interaction between PI3K/Akt and Smad3. Stimulation of skeletalmore » myoblasts with MSTN resulted in a transient increase in the pSmad3:Smad3 ratio and Smad-dependent transcription. Moreover, MSTN inhibited myod gene expression and myoblast fusion in an Activin receptor-like kinase/Smad3-dependent manner. Preincubation of skeletal myoblasts with IGF-1 blocked MSTN-induced Smad3 activation, promoting myod expression and myoblast differentiation. This inhibitory effect of IGF-1 on the MSTN signaling pathway was dependent on IGF-1 receptor, PI3K, and Akt activities. Finally, immunoprecipitation assay analysis determined that IGF-1 pretreatment increased Akt and Smad3 interaction. These results demonstrate that the IGF-1/PI3K/Akt pathway may inhibit MSTN signaling during myoblast differentiation, providing new insight to existing knowledge on the complex crosstalk between both growth factors. - Highlights: • IGF-1 inhibits Myostatin canonical signaling pathway through IGF-1R/PI3K/Akt pathway. • IGF-1 promotes myoblast differentiation through a direct blocking of Myostatin signaling pathway. • IGF-1 induces the interaction of Akt with Smad3 in skeletal myoblast.« less

Interferon-gamma regulates nucleoside transport systems in macrophages through signal transduction and activator of transduction factor 1 (STAT1)-dependent and -independent signalling pathways.

PubMed Central

Soler, Concepció; Felipe, Antonio; García-Manteiga, José; Serra, Maria; Guillén-Gómez, Elena; Casado, F Javier; MacLeod, Carol; Modolell, Manuel; Pastor-Anglada, Marçal; Celada, Antonio

2003-01-01

The expressions of CNT and ENT (concentrative and equilibrative nucleoside transporters) in macrophages are differentially regulated by IFN-gamma (interferon-gamma). This cytokine controls gene expression through STAT1-dependent and/or -independent pathways (where STAT1 stands for signal transduction and activator of transcription 1). In the present study, the role of STAT1 in the response of nucleoside transporters to IFN-gamma was studied using macrophages from STAT1 knockout mice. IFN-gamma triggered an inhibition of ENT1-related nucleoside transport activity through STAT1-dependent mechanisms. Such inhibition of macrophage growth and ENT1 activity by IFN-gamma is required for DNA synthesis. Interestingly, IFN-gamma led to an induction of the CNT1- and CNT2-related nucleoside transport activities independent of STAT1, thus ensuring the supply of extracellular nucleosides for the STAT1-independent RNA synthesis. IFN-gamma up-regulated CNT2 mRNA and CNT1 protein levels and down-regulated ENT1 mRNA in both wild-type and STAT1 knockout macrophages. This is consistent with a STAT1-independent, long-term-mediated, probably transcription-dependent, regulation of nucleoside transporter genes. Moreover, STAT1-dependent post-transcriptional mechanisms are implicated in the regulation of ENT1 activity. Although nitric oxide is involved in the regulation of ENT1 activity in B-cells at a post-transcriptional level, our results show that STAT1-dependent induction of nitric oxide by IFN-gamma is not implicated in the regulation of ENT1 activity in macrophages. Our results indicate that both STAT1-dependent and -independent pathways are involved in the regulation of nucleoside transporters by IFN-gamma in macrophages. PMID:12868960

Ubiquitin-Dependent Regulation of the Mammalian Hippo Pathway: Therapeutic Implications for Cancer

PubMed Central

Nguyen, Thanh Hung

2018-01-01

The Hippo pathway serves as a key barrier for oncogenic transformation. It acts by limiting the activity of the proto-oncogenes YAP and TAZ. Reduced Hippo signaling and elevated YAP/TAZ activities are frequently observed in various types of tumors. Emerging evidence suggests that the ubiquitin system plays an important role in regulating Hippo pathway activity. Deregulation of ubiquitin ligases and of deubiquitinating enzymes has been implicated in increased YAP/TAZ activity in cancer. In this article, we review recent insights into the ubiquitin-mediated regulation of the mammalian Hippo pathway, its deregulation in cancer, and possibilities for targeting the Hippo pathway through the ubiquitin system. PMID:29673168

Chronic administration of aripiprazole activates GSK3β-dependent signalling pathways, and up-regulates GABAA receptor expression and CREB1 activity in rats

PubMed Central

Pan, Bo; Huang, Xu-Feng; Deng, Chao

2016-01-01

Aripiprazole is a D2-like receptor (D2R) partial agonist with a favourable clinical profile. Previous investigations indicated that acute and short-term administration of aripiprazole had effects on PKA activity, GSK3β-dependent pathways, GABAA receptors, NMDA receptor and CREB1 in the brain. Since antipsychotics are used chronically in clinics, the present study investigated the long-term effects of chronic oral aripiprazole treatment on these cellular signalling pathways, in comparison with haloperidol (a D2R antagonist) and bifeprunox (a potent D2R partial agonist). We found that the Akt-GSK3β pathway was activated by aripiprazole and bifeprunox in the prefrontal cortex; NMDA NR2A levels were reduced by aripiprazole and haloperidol. In the nucleus accumbens, all three drugs increased Akt-GSK3β signalling; in addition, both aripiprazole and haloperidol, but not bifeprunox, increased the expression of Dvl-3, β-catenin and GABAA receptors, NMDA receptor subunits, as well as CREB1 phosphorylation levels. The results suggest that chronic oral administration of aripiprazole affects schizophrenia-related cellular signalling pathways and markers (including Akt-GSK3β signalling, Dvl-GSK3β-β-catenin signalling, GABAA receptor, NMDA receptor and CREB1) in a brain-region-dependent manner; the selective effects of aripiprazole on these signalling pathways might be associated with its unique clinical effects. PMID:27435909

The attenuated inflammation of MPL is due to the lack of CD14-dependent tight dimerization of the TLR4/MD2 complex at the plasma membrane.

PubMed

Tanimura, Natsuko; Saitoh, Shin-Ichiroh; Ohto, Umeharu; Akashi-Takamura, Sachiko; Fujimoto, Yukari; Fukase, Koichi; Shimizu, Toshiyuki; Miyake, Kensuke

2014-06-01

TLR4/MD-2 senses lipid A, activating the MyD88-signaling pathway on the plasma membrane and the TRIF-signaling pathway after CD14-mediated TLR4/MD-2 internalization into endosomes. Monophosphoryl lipid A (MPL), a detoxified derivative of lipid A, is weaker than lipid A in activating the MyD88-dependent pathway. Little is known, however, about mechanisms underlying the attenuated activation of MyD88-dependent pathways. We here show that MPL was impaired in induction of CD14-dependent TLR4/MD-2 dimerization compared with lipid A. Impaired TLR4/MD-2 dimerization decreased CD14-mediated TNFα production. In contrast, MPL was comparable to lipid A in CD14-independent MyD88-dependent TNFα production and TRIF-dependent responses including cell surface CD86 up-regulation and IFNβ induction. Although CD86 up-regulation is dependent on TRIF signaling, it was induced by TLR4/MD-2 at the plasma membrane. These results revealed that the attenuated MPL responses were due to CD14-initiated responses at the plasma membrane, but not just to responses initiated by MyD88, that is, MPL was specifically unable to induce CD14-dependent TLR4/MD-2 dimerization that selectively enhances MyD88-mediated responses at the plasma membrane. © The Japanese Society for Immunology. 2013. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Characterization of Puma-Dependent and Puma-Independent Neuronal Cell Death Pathways following Prolonged Proteasomal Inhibition▿

PubMed Central

Tuffy, Liam P.; Concannon, Caoimhín G.; D'Orsi, Beatrice; King, Matthew A.; Woods, Ina; Huber, Heinrich J.; Ward, Manus W.; Prehn, Jochen H. M.

2010-01-01

Proteasomal stress and the accumulation of polyubiquitinated proteins are key features of numerous neurodegenerative disorders. Previously we demonstrated that stabilization of p53 and activation of its target gene, puma (p53-upregulated mediator of apoptosis), mediated proteasome inhibitor-induced apoptosis in cancer cells. Here we demonstrated that Puma also contributed to proteasome inhibitor-induced apoptosis in mouse neocortical neurons. Although protection afforded by puma gene deletion was incomplete, we found little evidence indicating contributions from other proapoptotic BH3-only proteins. Attenuation of bax expression did not further reduce Puma-independent apoptosis, suggesting that pathways other than the mitochondrial apoptosis pathway were activated. Real-time imaging experiments in wild-type and puma-deficient neurons using a fluorescence resonance energy transfer (FRET)-based caspase sensor confirmed the involvement of a second cell death pathway characterized by caspase activation prior to mitochondrial permeabilization and, more prominently, a third, caspase-independent and Puma-independent pathway characterized by rapid cell shrinkage and nuclear condensation. This pathway involved lysosomal permeabilization in the absence of autophagy activation and was sensitive to cathepsin but not autophagy inhibition. Our data demonstrate that proteasomal stress activates distinct cell death pathways in neurons, leading to both caspase-dependent and caspase-independent apoptosis, and demonstrate independent roles for Puma and lysosomal permeabilization in this model. PMID:20921277

Dynamic regulation of VEGF-inducible genes by an ERK/ERG/p300 transcriptional network.

PubMed

Fish, Jason E; Cantu Gutierrez, Manuel; Dang, Lan T; Khyzha, Nadiya; Chen, Zhiqi; Veitch, Shawn; Cheng, Henry S; Khor, Melvin; Antounians, Lina; Njock, Makon-Sébastien; Boudreau, Emilie; Herman, Alexander M; Rhyner, Alexander M; Ruiz, Oscar E; Eisenhoffer, George T; Medina-Rivera, Alejandra; Wilson, Michael D; Wythe, Joshua D

2017-07-01

The transcriptional pathways activated downstream of vascular endothelial growth factor (VEGF) signaling during angiogenesis remain incompletely characterized. By assessing the signals responsible for induction of the Notch ligand delta-like 4 (DLL4) in endothelial cells, we find that activation of the MAPK/ERK pathway mirrors the rapid and dynamic induction of DLL4 transcription and that this pathway is required for DLL4 expression. Furthermore, VEGF/ERK signaling induces phosphorylation and activation of the ETS transcription factor ERG, a prerequisite for DLL4 induction. Transcription of DLL4 coincides with dynamic ERG-dependent recruitment of the transcriptional co-activator p300. Genome-wide gene expression profiling identified a network of VEGF-responsive and ERG-dependent genes, and ERG chromatin immunoprecipitation (ChIP)-seq revealed the presence of conserved ERG-bound putative enhancer elements near these target genes. Functional experiments performed in vitro and in vivo confirm that this network of genes requires ERK, ERG and p300 activity. Finally, genome-editing and transgenic approaches demonstrate that a highly conserved ERG-bound enhancer located upstream of HLX (which encodes a transcription factor implicated in sprouting angiogenesis) is required for its VEGF-mediated induction. Collectively, these findings elucidate a novel transcriptional pathway contributing to VEGF-dependent angiogenesis. © 2017. Published by The Company of Biologists Ltd.

Ral-Arf6 crosstalk regulates Ral dependent exocyst trafficking and anchorage independent growth signalling.

PubMed

Pawar, Archana; Meier, Jeremy A; Dasgupta, Anwesha; Diwanji, Neha; Deshpande, Neha; Saxena, Kritika; Buwa, Natasha; Inchanalkar, Siddhi; Schwartz, Martin Alexander; Balasubramanian, Nagaraj

2016-09-01

Integrin dependent regulation of growth factor signalling confers anchorage dependence that is deregulated in cancers. Downstream of integrins and oncogenic Ras the small GTPase Ral is a vital mediator of adhesion dependent trafficking and signalling. This study identifies a novel regulatory crosstalk between Ral and Arf6 that controls Ral function in cells. In re-adherent mouse fibroblasts (MEFs) integrin dependent activation of RalA drives Arf6 activation. Independent of adhesion constitutively active RalA and RalB could both however activate Arf6. This is further conserved in oncogenic H-Ras containing bladder cancer T24 cells, which express anchorage independent active Ral that supports Arf6 activation. Arf6 mediates active Ral-exocyst dependent delivery of raft microdomains to the plasma membrane that supports anchorage independent growth signalling. Accordingly in T24 cells the RalB-Arf6 crosstalk is seen to preferentially regulate anchorage independent Erk signalling. Active Ral we further find uses a Ral-RalBP1-ARNO-Arf6 pathway to mediate Arf6 activation. This study hence identifies Arf6, through this regulatory crosstalk, to be a key downstream mediator of Ral isoform function along adhesion dependent pathways in normal and cancer cells. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Hippo signaling promotes JNK-dependent cell migration.

PubMed

Ma, Xianjue; Wang, Hongxiang; Ji, Jiansong; Xu, Wenyan; Sun, Yihao; Li, Wenzhe; Zhang, Xiaoping; Chen, Juxiang; Xue, Lei

2017-02-21

Overwhelming studies show that dysregulation of the Hippo pathway is positively correlated with cell proliferation, growth, and tumorigenesis. Paradoxically, the detailed molecular roles of the Hippo pathway in cell invasion remain debatable. Using a Drosophila invasion model in wing epithelium, we show herein that activated Hippo signaling promotes cell invasion and epithelial-mesenchymal transition through JNK, as inhibition of JNK signaling dramatically blocked Hippo pathway activation-induced matrix metalloproteinase 1 expression and cell invasion. Furthermore, we identify bantam -Rox8 modules as essential components downstream of Yorkie in mediating JNK-dependent cell invasion. Finally, we confirm that YAP (Yes-associated protein) expression negatively regulates TIA1 (Rox8 ortholog) expression and cell invasion in human cancer cells. Together, these findings provide molecular insights into Hippo pathway-mediated cell invasion and also raise a noteworthy concern in therapeutic interventions of Hippo-related cancers, as simply inhibiting Yorkie or YAP activity might paradoxically accelerate cell invasion and metastasis.

A Comparison of Blood Factor XII Autoactivation in Buffer, Protein Cocktail, Serum, and Plasma Solutions

PubMed Central

Golas, Avantika; Yeh, Chyi-Huey Josh; Pitakjakpipop, Harit; Siedlecki, Christopher A.; Vogler, Erwin A.

2012-01-01

Activation of blood plasma coagulation in vitro by contact with material surfaces is demonstrably dependent on plasma-volume-to-activator-surface-area ratio. The only plausible explanation consistent with current understanding of coagulation-cascade biochemistry is that procoagulant stimulus arising from the activation complex of the intrinsic pathway is dependent on activator surface area. And yet, it is herein shown that activation of the blood zymogen factor XII (Hageman factor, FXII) dissolved in buffer, protein cocktail, heat-denatured serum, and FXI deficient plasma does not exhibit activator surface-area dependence. Instead, a highly-variable burst of procoagulant-enzyme yield is measured that exhibits no measurable kinetics, sensitivity to mixing, or solution-temperature dependence. Thus, FXII activation in both buffer and protein-containing solutions does not exhibit characteristics of a biochemical reaction but rather appears to be a “mechanochemical” reaction induced by FXII molecule interactions with hydrophilic activator particles that do not formally adsorb blood proteins from solution. Results of this study strongly suggest that activator surface-area dependence observed in contact activation of plasma coagulation does not solely arise at the FXII activation step of the intrinsic pathway. PMID:23117212

Nucleolus-derived mediators in oncogenic stress response and activation of p53-dependent pathways.

PubMed

Stępiński, Dariusz

2016-08-01

Rapid growth and division of cells, including tumor ones, is correlated with intensive protein biosynthesis. The output of nucleoli, organelles where translational machineries are formed, depends on a rate of particular stages of ribosome production and on accessibility of elements crucial for their effective functioning, including substrates, enzymes as well as energy resources. Different factors that induce cellular stress also often lead to nucleolar dysfunction which results in ribosome biogenesis impairment. Such nucleolar disorders, called nucleolar or ribosomal stress, usually affect cellular functioning which in fact is a result of p53-dependent pathway activation, elicited as a response to stress. These pathways direct cells to new destinations such as cell cycle arrest, damage repair, differentiation, autophagy, programmed cell death or aging. In the case of impaired nucleolar functioning, nucleolar and ribosomal proteins mediate activation of the p53 pathways. They are also triggered as a response to oncogenic factor overexpression to protect tissues and organs against extensive proliferation of abnormal cells. Intentional impairment of any step of ribosome biosynthesis which would direct the cells to these destinations could be a strategy used in anticancer therapy. This review presents current knowledge on a nucleolus, mainly in relation to cancer biology, which is an important and extremely sensitive element of the mechanism participating in cellular stress reaction mediating activation of the p53 pathways in order to counteract stress effects, especially cancer development.

The E3 ubiquitin ligase mind bomb-2 (MIB2) protein controls B-cell CLL/lymphoma 10 (BCL10)-dependent NF-κB activation.

PubMed

Stempin, Cinthia C; Chi, Liying; Giraldo-Vela, Juan P; High, Anthony A; Häcker, Hans; Redecke, Vanessa

2011-10-28

B-cell CLL/lymphoma 10 (BCL10) is crucial for the activation of NF-κB in numerous immune receptor signaling pathways, including the T-cell receptor (TCR) and B-cell receptor signaling pathways. However, the molecular mechanisms that lead to signal transduction from BCL10 to downstream NF-κB effector kinases, such as TAK1 and components of the IKK complex, are not entirely understood. Here we used a proteomic approach and identified the E3 ligase MIB2 as a novel component of the activated BCL10 complex. In vitro translation and pulldown assays suggest direct interaction between BCL10 and MIB2. Overexpression experiments show that MIB2 controls BCL10-mediated activation of NF-κB by promoting autoubiquitination and ubiquitination of IKKγ/NEMO, as well as recruitment and activation of TAK1. Knockdown of MIB2 inhibited BCL10-dependent NF-κB activation. Together, our results identify MIB2 as a novel component of the activated BCL10 signaling complex and a missing link in the BCL10-dependent NF-κB signaling pathway.

DAF-16-dependent suppression of immunity during reproduction in Caenorhabditis elegans.

PubMed

Miyata, Sachiko; Begun, Jakob; Troemel, Emily R; Ausubel, Frederick M

2008-02-01

To further understand how the nematode Caenorhabditis elegans defends itself against pathogen attack, we analyzed enhanced pathogen resistance (epr) mutants obtained from a forward genetic screen. We also examined several well-characterized sterile mutants that exhibit an Epr phenotype. We found that sterility and pathogen resistance are highly correlated and that resistance in both epr and sterile mutants is dependent on DAF-16 activity. Our data indicate that a DAF-16-dependent signaling pathway distinct from previously described pathways is involved in the activation of genes that confer resistance to bacterial pathogens. The timing of DAF-16-dependent gene activation in sterile mutants coincides with the onset of embryonic development in wild-type animals, suggesting that signals from developing embryos normally downregulate the immune response.

Modeling Protein Expression and Protein Signaling Pathways

PubMed Central

Telesca, Donatello; Müller, Peter; Kornblau, Steven M.; Suchard, Marc A.; Ji, Yuan

2015-01-01

High-throughput functional proteomic technologies provide a way to quantify the expression of proteins of interest. Statistical inference centers on identifying the activation state of proteins and their patterns of molecular interaction formalized as dependence structure. Inference on dependence structure is particularly important when proteins are selected because they are part of a common molecular pathway. In that case, inference on dependence structure reveals properties of the underlying pathway. We propose a probability model that represents molecular interactions at the level of hidden binary latent variables that can be interpreted as indicators for active versus inactive states of the proteins. The proposed approach exploits available expert knowledge about the target pathway to define an informative prior on the hidden conditional dependence structure. An important feature of this prior is that it provides an instrument to explicitly anchor the model space to a set of interactions of interest, favoring a local search approach to model determination. We apply our model to reverse-phase protein array data from a study on acute myeloid leukemia. Our inference identifies relevant subpathways in relation to the unfolding of the biological process under study. PMID:26246646

Differential PKA activation and AKAP association determines cell fate in cancer cells

PubMed Central

2013-01-01

Background The dependence of malignant properties of colorectal cancer (CRC) cells on IGF1R signaling has been demonstrated and several IGF1R antagonists are currently in clinical trials. Recently, we identified a novel pathway in which cAMP independent PKA activation by TGFβ signaling resulted in the destabilization of survivin/XIAP complex leading to increased cell death. In this study, we evaluated the effect of IGF1R inhibition or activation on PKA activation and its downstream cell survival signaling mechanisms. Methods Small molecule IGF1R kinase inhibitor OSI-906 was used to test the effect of IGF1R inhibition on PKA activation, AKAP association and its downstream cell survival signaling. In a complementary approach, ligand mediated activation of IGF1R was performed and AKAP/PKA signaling was analyzed for their downstream survival effects. Results We demonstrate that the inhibition of IGF1R in the IGF1R-dependent CRC subset generates cell death through a novel mechanism involving TGFβ stimulated cAMP independent PKA activity that leads to disruption of cell survival by survivin/XIAP mediated inhibition of caspase activity. Importantly, ligand mediated activation of the IGF1R in CRC cells results in the generation of cAMP dependent PKA activity that functions in cell survival by inhibiting caspase activity. Therefore, this subset of CRC demonstrates 2 opposing pathways organized by 2 different AKAPs in the cytoplasm that both utilize activation of PKA in a manner that leads to different outcomes with respect to life and death. The cAMP independent PKA activation pathway is dependent upon mitochondrial AKAP149 for its apoptotic functions. In contrast, Praja2 (Pja2), an AKAP-like E3 ligase protein was identified as a key element in controlling cAMP dependent PKA activity and pro-survival signaling. Genetic manipulation of AKAP149 and Praja2 using siRNA KD had opposing effects on PKA activity and survivin/XIAP regulation. Conclusions We had identified 2 cytoplasmic pathways dependent upon the same enzymatic activity with opposite effects on cell fate in terms of life and death. Understanding the specific mechanistic functions of IGF1R with respect to determining the PKA survival functions would have potential for impact upon the development of new therapeutic strategies by exploiting the IGF1R/cAMP-PKA survival signaling in cancer. PMID:24083380

Drosophila insulin and target of rapamycin (TOR) pathways regulate GSK3 beta activity to control Myc stability and determine Myc expression in vivo.

PubMed

Parisi, Federica; Riccardo, Sara; Daniel, Margaret; Saqcena, Mahesh; Kundu, Nandini; Pession, Annalisa; Grifoni, Daniela; Stocker, Hugo; Tabak, Esteban; Bellosta, Paola

2011-09-27

Genetic studies in Drosophila melanogaster reveal an important role for Myc in controlling growth. Similar studies have also shown how components of the insulin and target of rapamycin (TOR) pathways are key regulators of growth. Despite a few suggestions that Myc transcriptional activity lies downstream of these pathways, a molecular mechanism linking these signaling pathways to Myc has not been clearly described. Using biochemical and genetic approaches we tried to identify novel mechanisms that control Myc activity upon activation of insulin and TOR signaling pathways. Our biochemical studies show that insulin induces Myc protein accumulation in Drosophila S2 cells, which correlates with a decrease in the activity of glycogen synthase kinase 3-beta (GSK3β ) a kinase that is responsible for Myc protein degradation. Induction of Myc by insulin is inhibited by the presence of the TOR inhibitor rapamycin, suggesting that insulin-induced Myc protein accumulation depends on the activation of TOR complex 1. Treatment with amino acids that directly activate the TOR pathway results in Myc protein accumulation, which also depends on the ability of S6K kinase to inhibit GSK3β activity. Myc upregulation by insulin and TOR pathways is a mechanism conserved in cells from the wing imaginal disc, where expression of Dp110 and Rheb also induces Myc protein accumulation, while inhibition of insulin and TOR pathways result in the opposite effect. Our functional analysis, aimed at quantifying the relative contribution of Myc to ommatidial growth downstream of insulin and TOR pathways, revealed that Myc activity is necessary to sustain the proliferation of cells from the ommatidia upon Dp110 expression, while its contribution downstream of TOR is significant to control the size of the ommatidia. Our study presents novel evidence that Myc activity acts downstream of insulin and TOR pathways to control growth in Drosophila. At the biochemical level we found that both these pathways converge at GSK3β to control Myc protein stability, while our genetic analysis shows that insulin and TOR pathways have different requirements for Myc activity during development of the eye, suggesting that Myc might be differentially induced by these pathways during growth or proliferation of cells that make up the ommatidia.

Constitutive activation of jasmonate signaling in an Arabidopsis mutant correlates with enhanced resistance to Erysiphe cichoracearum, Pseudomonas syringae, and Myzus persicae.

PubMed

Ellis, Christine; Karafyllidis, Ioannis; Turner, John G

2002-10-01

In Arabidopsis spp., the jasmonate (JA) response pathway generally is required for defenses against necrotrophic pathogens and chewing insects, while the salicylic acid (SA) response pathway is generally required for specific, resistance (R) gene-mediated defenses against both biotrophic and necrotrophic pathogens. For example, SA-dependent defenses are required for resistance to the biotrophic fungal pathogen Erysiphe cichoracearum UCSC1 and the bacterial pathogen Pseudomonas syringae pv. maculicola, and also are expressed during response to the green peach aphid Myzus persicae. However, recent evidence indicates that the expression of JA-dependent defenses also may confer resistance to E. cichoracearum. To confirm and to extend this observation, we have compared the disease and pest resistance of wild-type Arabidopsis plants with that of the mutants coil, which is insensitive to JA, and cev1, which has constitutive JA signaling. Measurements of the colonization of these plants by E. cichoracearum, P. syringae pv. maculicola, and M. persicae indicated that activation of the JA signal pathway enhanced resistance, and was associated with the activation of JA-dependent defense genes and the suppression of SA-dependent defense genes. We conclude that JA and SA induce alternative defense pathways that can confer resistance to the same pathogens and pests.

Activity-Dependent Bidirectional Regulation of GAD Expression in a Homeostatic Fashion Is Mediated by BDNF-Dependent and Independent Pathways

PubMed Central

Hanno-Iijima, Yoko; Tanaka, Masami; Iijima, Takatoshi

2015-01-01

Homeostatic synaptic plasticity, or synaptic scaling, is a mechanism that tunes neuronal transmission to compensate for prolonged, excessive changes in neuronal activity. Both excitatory and inhibitory neurons undergo homeostatic changes based on synaptic transmission strength, which could effectively contribute to a fine-tuning of circuit activity. However, gene regulation that underlies homeostatic synaptic plasticity in GABAergic (GABA, gamma aminobutyric) neurons is still poorly understood. The present study demonstrated activity-dependent dynamic scaling in which NMDA-R (N-methyl-D-aspartic acid receptor) activity regulated the expression of GABA synthetic enzymes: glutamic acid decarboxylase 65 and 67 (GAD65 and GAD67). Results revealed that activity-regulated BDNF (brain-derived neurotrophic factor) release is necessary, but not sufficient, for activity-dependent up-scaling of these GAD isoforms. Bidirectional forms of activity-dependent GAD expression require both BDNF-dependent and BDNF-independent pathways, both triggered by NMDA-R activity. Additional results indicated that these two GAD genes differ in their responsiveness to chronic changes in neuronal activity, which could be partially caused by differential dependence on BDNF. In parallel to activity-dependent bidirectional scaling in GAD expression, the present study further observed that a chronic change in neuronal activity leads to an alteration in neurotransmitter release from GABAergic neurons in a homeostatic, bidirectional fashion. Therefore, the differential expression of GAD65 and 67 during prolonged changes in neuronal activity may be implicated in some aspects of bidirectional homeostatic plasticity within mature GABAergic presynapses. PMID:26241953

Activity-Dependent Bidirectional Regulation of GAD Expression in a Homeostatic Fashion Is Mediated by BDNF-Dependent and Independent Pathways.

PubMed

Hanno-Iijima, Yoko; Tanaka, Masami; Iijima, Takatoshi

2015-01-01

Homeostatic synaptic plasticity, or synaptic scaling, is a mechanism that tunes neuronal transmission to compensate for prolonged, excessive changes in neuronal activity. Both excitatory and inhibitory neurons undergo homeostatic changes based on synaptic transmission strength, which could effectively contribute to a fine-tuning of circuit activity. However, gene regulation that underlies homeostatic synaptic plasticity in GABAergic (GABA, gamma aminobutyric) neurons is still poorly understood. The present study demonstrated activity-dependent dynamic scaling in which NMDA-R (N-methyl-D-aspartic acid receptor) activity regulated the expression of GABA synthetic enzymes: glutamic acid decarboxylase 65 and 67 (GAD65 and GAD67). Results revealed that activity-regulated BDNF (brain-derived neurotrophic factor) release is necessary, but not sufficient, for activity-dependent up-scaling of these GAD isoforms. Bidirectional forms of activity-dependent GAD expression require both BDNF-dependent and BDNF-independent pathways, both triggered by NMDA-R activity. Additional results indicated that these two GAD genes differ in their responsiveness to chronic changes in neuronal activity, which could be partially caused by differential dependence on BDNF. In parallel to activity-dependent bidirectional scaling in GAD expression, the present study further observed that a chronic change in neuronal activity leads to an alteration in neurotransmitter release from GABAergic neurons in a homeostatic, bidirectional fashion. Therefore, the differential expression of GAD65 and 67 during prolonged changes in neuronal activity may be implicated in some aspects of bidirectional homeostatic plasticity within mature GABAergic presynapses.

Requirement for Pectin Methyl Esterase and Preference for Fragmented over Native Pectins for Wall-associated Kinase-activated, EDS1/PAD4-dependent Stress Response in Arabidopsis*

PubMed Central

Kohorn, Bruce D.; Kohorn, Susan L.; Saba, Nicholas J.; Martinez, Victoriano Meco

2014-01-01

The wall-associated kinases (WAKs) have a cytoplasmic protein kinase domain that spans the plasma membrane and binds pectin in the extracellular matrix of plants. WAKs are required for cell expansion during Arabidopsis seedling development but are also an integral part of the response to pathogens and stress that present oligogalacturonides (OGs), which subsequently bind to WAKs and activate a MPK6 (mitogen-activated protein kinase)-dependent pathway. It was unclear how WAKs distinguish native pectin polymers and OGs to activate one or the other of these two pathways. A dominant allele of WAK2 constitutively activates the stress response, and we show here that the effect is dependent upon EDS1 and PAD4, transcriptional activators involved in the pathogen response. Moreover, the WAK2 dominant allele is suppressed by a null allele of a pectin methyl esterase (PME3) whose activity normally leads to cross-linking of pectins in the cell wall. Although OGs activate a transcriptional response in wild type, the response is enhanced in a pme3/pme3 null, consistent with a competition by OG and native polymers for activation of WAKs. This provides a plausible mechanism for WAKs to distinguish an expansion from a stress pathway. PMID:24855660

Voltage-dependent neuromodulation of Na+ channels by D1-like dopamine receptors in rat hippocampal neurons.

PubMed

Cantrell, A R; Scheuer, T; Catterall, W A

1999-07-01

Activation of D1-like dopamine (DA) receptors reduces peak Na+ current in acutely isolated hippocampal neurons through phosphorylation of the alpha subunit of the Na+ channel by cAMP-dependent protein kinase (PKA). Here we report that neuromodulation of Na+ currents by DA receptors via PKA is voltage-dependent in the range of -110 to -70 mV and is also sensitive to concurrent activation of protein kinase C (PKC). Depolarization enhanced the ability of D1-like DA receptors to reduce peak Na+ currents via the PKA pathway. Similar voltage-dependent modulation was observed when PKA was activated directly with the membrane-permeant PKA activator DCl-cBIMPS (cBIMPS; 20 microM), indicating that the membrane potential dependence occurs downstream of PKA. PKA activation caused only a small (-2.9 mV) shift in the voltage dependence of steady-state inactivation and had no effect on slow inactivation or on the rates of entry into the fast or slow inactivated states, suggesting that another mechanism is responsible for coupling of membrane potential changes to PKA modulation. Activation of PKC with a low concentration of the membrane-permeant diacylglycerol analog oleylacetyl glycerol also potentiated modulation by SKF 81297 or cBIMPS, and these effects were most striking at hyperpolarized membrane potentials where PKA modulation was not stimulated by membrane depolarization. Thus, activation of D1-like DA receptors causes a strong reduction in Na+ current via the PKA pathway, but it is effective primarily when it is combined with depolarization or activation of PKC. The convergence of these three distinct signaling modalities on the Na+ channel provides an intriguing mechanism for integration of information from multiple signaling pathways in the hippocampus and CNS.

Co-factors Required for TLR7- and TLR9- dependent Innate Immune Responses

PubMed Central

Chiang, Chih-yuan; Engel, Alex; Opaluch, Amanda M.; Ramos, Irene; Maestre, Ana M.; Secundino, Ismael; De Jesus, Paul D.; Nguyen, Quy T.; Welch, Genevieve; Bonamy, Ghislain M.C.; Miraglia, Loren J.; Orth, Anthony P.; Nizet, Victor; Fernandez-Sesma, Ana; Zhou, Yingyao; Barton, Gregory M.; Chanda, Sumit K.

2012-01-01

SUMMARY Pathogens commonly utilize endocytic pathways to gain cellular access. The endosomal pattern recognition receptors TLR7 and TLR9 detect pathogen-encoded nucleic acids to initiate MyD88-dependent pro-inflammatory responses to microbial infection. Using genome-wide RNAi screening and integrative systems-based analysis we identify 190 co-factors required for TLR7- and TLR9-directed signaling responses. A set of co-factors were cross-profiled for their activities downstream of several immunoreceptors, and then functionally mapped based on the known architecture of NF-κB signaling pathways. Protein complexes and pathways involved in ubiquitin-protein ligase activities, sphingolipid metabolism, chromatin modifications, and ancient stress responses were found to modulate innate recognition of endosomal nucleic acids. Additionally, hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) was characterized as necessary for ubiquitin-dependent TLR9 targeting to the endolysosome. Proteins and pathways identified here should prove useful in delineating strategies to manipulate innate responses for treatment of autoimmune disorders and microbial infection. PMID:22423970

JAK2-V617F-induced MAPK activity is regulated by PI3K and acts synergistically with PI3K on the proliferation of JAK2-V617F-positive cells

PubMed Central

Wolf, Alexandra; Eulenfeld, René; Gäbler, Karoline; Rolvering, Catherine; Haan, Serge; Behrmann, Iris; Denecke, Bernd; Haan, Claude; Schaper, Fred

2013-01-01

The identification of a constitutively active JAK2 mutant, namely JAK2-V617F, was a milestone in the understanding of Philadelphia chromosome-negative myeloproliferative neoplasms. The JAK2-V617F mutation confers cytokine hypersensitivity, constitutive activation of the JAK-STAT pathway, and cytokine-independent growth. In this study we investigated the mechanism of JAK2-V617F-dependent signaling with a special focus on the activation of the MAPK pathway. We observed JAK2-V617F-dependent deregulated activation of the multi-site docking protein Gab1 as indicated by constitutive, PI3K-dependent membrane localization and tyrosine phosphorylation of Gab1. Furthermore, we demonstrate that PI3K signaling regulates MAPK activation in JAK2-V617F-positve cells. This cross-regulation of the MAPK pathway by PI3K affects JAK2-V617F-specific target gene induction, erythroid colony formation, and regulates proliferation of JAK2-V617F-positive patient cells in a synergistically manner. PMID:24069558

cPLA2α Gene Activation by IL-1β is Dependent on an Upstream Kinase pathway, Enzymatic Activation and Downstream 15-lipoxygenase Activity: A Positive Feedback Loop

PubMed Central

Walters, Jewell N.; Bickford, Justin S.; Beachy, Dawn E.; Newsom, Kimberly J.; Herlihy, John-David H.; Peck, Molly V.; Qiu, Xiaolei; Nick, Harry S.

2011-01-01

Cytosolic phospholipase A2α (cPLA2α) is the most widely studied member of the Group IV PLA2 family. The enzyme is Ca2+-dependent with specificity for phospholipid substrates containing arachidonic acid. As the pinnacle of the arachidonic acid pathway, cPLA2α has a primary role in the biosynthesis of a diverse family of eicosanoid metabolites, with potent physiological, inflammatory and pathological consequences. cPLA2α activity is regulated by pro-inflammatory stimuli through pathways involving increased intracellular Ca2+ levels, phosphorylation coupled to increased enzymatic activity and de novo gene transcription. This study addresses the signal transduction pathways for protein phosphorylation and gene induction following IL-1β stimulation in human fetal lung fibroblasts. Our results utilizing both inhibitors and kinase-deficient cells demonstrate that cPLA2α is phosphorylated within 10 min of IL-1β treatment, an event requiring p38 MAPK as well as the upstream kinase, MKK3/MKK6. Inhibition of p38 MAPK also blocks the phosphorylation of a downstream, nuclear kinase, MSK-1. Our results further demonstrate that the activities of both cPLA2α and a downstream lipoxygenase (15-LOX2) are required for IL-1β-dependent induction of cPLA2α mRNA expression. Overall, these data support an MKK3/MKK6→p38 MAPK→MSK-1→cPLA2α→15-LOX2-dependent, positive feedback loop where a protein’s enzymatic activity is required to regulate its own gene induction by a pro-inflammatory stimulus. PMID:21771656

Use Dependence of Heat Sensitivity of Vanilloid Receptor TRPV2.

PubMed

Liu, Beiying; Qin, Feng

2016-04-12

Thermal TRP channels mediate temperature transduction and pain sensation. The vanilloid receptor TRPV2 is involved in detection of noxious heat in a subpopulation of high-threshold nociceptors. It also plays a critical role in development of thermal hyperalgesia, but the underlying mechanism remains uncertain. Here we analyze the heat sensitivity of the TRPV2 channel. Heat activation of the channel exhibits strong use dependence. Prior heat activation can profoundly alter its subsequent temperature responsiveness, causing decreases in both temperature activation threshold and slope sensitivity of temperature dependence while accelerating activation time courses. Notably, heat and agonist activations differ in cross use-dependence. Prior heat stimulation can dramatically sensitize agonist responses, but not conversely. Quantitative analyses indicate that the use dependence in heat sensitivity is pertinent to the process of temperature sensing by the channel. The use dependence of TRPV2 reveals that the channel can have a dynamic temperature sensitivity. The temperature sensing structures within the channel have multiple conformations and the temperature activation pathway is separate from the agonist activation pathway. Physiologically, the use dependence of TRPV2 confers nociceptors with a hypersensitivity to heat and thus provides a mechanism for peripheral thermal hyperalgesia. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Bladder volume-dependent excitatory and inhibitory influence of lumbosacral dorsal and ventral roots on bladder activity in rats

PubMed Central

Sugaya, Kimio; de Groat, William C.

2011-01-01

This study was undertaken to examine the role of the afferent and efferent pathways of the lumbosacral spinal nerve roots in the tonic control of bladder activity. Changes of isovolumetric bladder activity were recorded in 21 sympathectomized female rats under urethane anesthesia following transection of the dorsal (DRT) and ventral (VRT) lumbosacral spinal roots, and after intraperitoneal administration of hexamethonium. DRT altered the baseline intravesical pressure in a bladder volume-dependent manner in each animal. The percent change of baseline pressure after VRT following DRT was also dependent upon bladder volume. The percent change of baseline pressure after VRT alone was similarly dependent on bladder volume, but not after VRT followed by DRT. The percent change of baseline intravesical pressure (y)(−9 to +8 cm H2O, −56 to +46%) after DRT and VRT depended upon bladder volume (x)(y = 44.7 x −40.4) in all rats. Hexamethonium increased the amplitude of small myogenic bladder contractions after DRT and VRT. In conclusion, the bladder is tonically excited or inhibited by a local reflex pathway and by a parasympathetic reflex pathway that depends on connections with the lumbosacral spinal cord and the pelvic nerves. Both reflex mechanisms are influenced by bladder volume. PMID:17878597

A PLC-γ1-independent, RasGRP1-ERK dependent pathway drives lymphoproliferative disease in LAT-Y136F mutant mice

PubMed Central

Kortum, Robert L.; Rouquette-Jazdanian, Alexandre K.; Miyaji, Michihiko; Merrill, Robert K.; Markegard, Evan; Pinski, John M.; Wesselink, Amelia; Nath, Nandan N.; Alexander, Clayton P.; Li, Wenmei; Kedei, Noemi; Roose, Jeroen P.; Blumberg, Peter M.; Samelson, Lawrence E.; Sommers, Connie L.

2012-01-01

Mice expressing a germline mutation in the PLC-γ1 binding site of LAT (linker for activation of T cells) show progressive lymphoproliferation and ultimately die at 4–6 months of age. The hyper-activated T cells in these mice show defective TCR-induced calcium flux, but enhanced Ras/ERK activation that is critical for disease progression. Despite the loss of LAT-dependent PLC-γ1 binding and activation, genetic analysis revealed RasGRP1, and not Sos1 or Sos2, to be the major RasGEF responsible for ERK activation and the lymphoproliferative phenotype in these mice. Analysis of isolated CD4+ T cells from LAT-Y136F mice showed altered proximal TCR-dependent kinase signaling, which activated a Zap70- and LAT-independent pathway. Moreover, LAT-Y136F T cells showed ERK activation that was dependent on Lck and/or Fyn, PKCθ, and RasGRP1. These data demonstrate a novel route to Ras activation in vivo in a pathological setting. PMID:23209318

Dynamic imaging of adaptive stress response pathway activation for prediction of drug induced liver injury.

PubMed

Wink, Steven; Hiemstra, Steven W; Huppelschoten, Suzanne; Klip, Janna E; van de Water, Bob

2018-05-01

Drug-induced liver injury remains a concern during drug treatment and development. There is an urgent need for improved mechanistic understanding and prediction of DILI liabilities using in vitro approaches. We have established and characterized a panel of liver cell models containing mechanism-based fluorescent protein toxicity pathway reporters to quantitatively assess the dynamics of cellular stress response pathway activation at the single cell level using automated live cell imaging. We have systematically evaluated the application of four key adaptive stress pathway reporters for the prediction of DILI liability: SRXN1-GFP (oxidative stress), CHOP-GFP (ER stress/UPR response), p21 (p53-mediated DNA damage-related response) and ICAM1 (NF-κB-mediated inflammatory signaling). 118 FDA-labeled drugs in five human exposure relevant concentrations were evaluated for reporter activation using live cell confocal imaging. Quantitative data analysis revealed activation of single or multiple reporters by most drugs in a concentration and time dependent manner. Hierarchical clustering of time course dynamics and refined single cell analysis allowed the allusion of key events in DILI liability. Concentration response modeling was performed to calculate benchmark concentrations (BMCs). Extracted temporal dynamic parameters and BMCs were used to assess the predictive power of sub-lethal adaptive stress pathway activation. Although cellular adaptive responses were activated by non-DILI and severe-DILI compounds alike, dynamic behavior and lower BMCs of pathway activation were sufficiently distinct between these compound classes. The high-level detailed temporal- and concentration-dependent evaluation of the dynamics of adaptive stress pathway activation adds to the overall understanding and prediction of drug-induced liver liabilities.

A forward chemical genetic screen reveals an inhibitor of the Mre11–Rad50–Nbs1 complex

PubMed Central

Dupré, Aude; Boyer-Chatenet, Louise; Sattler, Rose M; Modi, Ami P; Lee, Ji-Hoon; Nicolette, Matthew L; Kopelovich, Levy; Jasin, Maria; Baer, Richard; Paull, Tanya T; Gautier, Jean

2009-01-01

The MRN (Mre11-Rad50-Nbs1)-ATM (ataxia-telangiectasia mutated) pathway is essential for sensing and signaling from DNA double-strand breaks. The MRN complex acts as a DNA damage sensor, maintains genome stability during DNA replication, promotes homology-dependent DNA repair and activates ATM. MRN is essential for cell viability, which has limited functional studies of the complex. Small-molecule inhibitors of MRN could circumvent this experimental limitation and could also be used as cellular radio- and chemosensitization compounds. Using cell-free systems that recapitulate faithfully the MRN-ATM signaling pathway, we designed a forward chemical genetic screen to identify inhibitors of the pathway, and we isolated Z-5-(4-hydroxybenzylidene)-2-imino-1,3-thiazolidin-4-one (mirin, 1) as an inhibitor of MRN. Mirin prevents MRN-dependent activation of ATM without affecting ATM protein kinase activity, and it inhibits Mre11-associated exonuclease activity. Consistent with its ability to target the MRN complex, mirin abolishes the G2/M checkpoint and homology-dependent repair in mammalian cells. PMID:18176557

Alternative Pathway of Metronidazole Activation in Trichomonas vaginalis Hydrogenosomes

PubMed Central

Hrdý, Ivan; Cammack, Richard; Stopka, Pavel; Kulda, Jaroslav; Tachezy, Jan

2005-01-01

Metronidazole and related 5-nitroimidazoles are the only available drugs in the treatment of human urogenital trichomoniasis caused by the protozoan parasite Trichomonas vaginalis. The drugs are activated to cytotoxic anion radicals by their reduction within the hydrogenosomes. It has been established that electrons required for metronidazole activation are released from pyruvate by the activity of pyruvate:ferredoxin oxidoreductase and transferred to the drug by a low-redox-potential carrier, ferredoxin. Here we describe a novel pathway involved in the drug activation within the hydrogenosome. The source of electrons is malate, another major hydrogenosomal substrate, which is oxidatively decarboxylated to pyruvate and CO2 by NAD-dependent malic enzyme. The electrons released during this reaction are transferred from NADH to ferredoxin by NADH dehydrogenase homologous to the catalytic module of mitochondrial complex I, which uses ferredoxin as electron acceptor. Trichomonads acquire high-level metronidazole resistance only after both pyruvate- and malate-dependent pathways of metronidazole activation are eliminated from the hydrogenosomes. PMID:16304169

Necrotrophic pathogens use the salicylic acid signaling pathway to promote disease development in tomato.

PubMed

Rahman, Taha Abd El; Oirdi, Mohamed El; Gonzalez-Lamothe, Rocio; Bouarab, Kamal

2012-12-01

Plants use different immune pathways to combat pathogens. The activation of the jasmonic acid (JA)-signaling pathway is required for resistance against necrotrophic pathogens; however, to combat biotrophic pathogens, the plants activate mainly the salicylic acid (SA)-signaling pathway. SA can antagonize JA signaling and vice versa. NPR1 (noninducible pathogenesis-related 1) is considered a master regulator of SA signaling. NPR1 interacts with TGA transcription factors, ultimately leading to the activation of SA-dependent responses. SA has been shown to promote disease development caused by the necrotrophic pathogen Botrytis cinerea through NPR1, by suppressing the expression of two JA-dependent defense genes, proteinase inhibitors I and II. We show here that the transcription factor TGA1.a contributes to disease development caused by B. cinerea in tomato by suppressing the expression of proteinase inhibitors I and II. Finally, we present evidence that the SA-signaling pathway contributes to disease development caused by another necrotrophic pathogen, Alternaria solani, in tomato. Disease development promoted by SA through NPR1 requires the TGA1.a transcription factor. These data highlight how necrotrophs manipulate the SAsignaling pathway to promote their disease in tomato.

Depletion of ribosomal protein L37 occurs in response to DNA damage and activates p53 through the L11/MDM2 pathway.

PubMed

Llanos, Susana; Serrano, Manuel

2010-10-01

Perturbation of ribosomal biogenesis has recently emerged as a relevant p53-activating pathway. This pathway can be initiated by depletion of certain ribosomal proteins, which is followed by the binding and inhibition of MDM2 by a different subset of ribosomal proteins that includes L11. Here, we report that depletion of L37 leads to cell cycle arrest in a L11- and p53-dependent manner. DNA damage can initiate ribosomal stress, although little is known about the mechanisms involved. We have found that some genotoxic insults, namely, UV light and cisplatin, lead to proteasomal degradation of L37 in the nucleoplasm and to the ensuing L11-dependent stabilization of p53. Moreover, ectopic L37 overexpression can attenuate the DNA damage response mediated by p53. These results support the concept that DNA damage-induced proteasomal degradation of L37 constitutes a mechanistic link between DNA damage and the ribosomal stress pathway, and is a relevant contributing signaling pathway for the activation of p53 in response to DNA damage.

Activity-induced convergence of APP and BACE-1 in acidic microdomains via an endocytosis-dependent pathway

PubMed Central

Das, Utpal; Scott, David; Ganguly, Archan; Koo, Edward H.; Tang, Yong; Roy, Subhojit

2013-01-01

The convergence of APP (substrate) and BACE-1 (enzyme) is a rate-limiting, obligatory event triggering the amyloidogenic pathway – a key step in Alzheimer’s disease (AD) pathology. However, as both APP/BACE-1 are highly expressed in brain, mechanisms precluding their unabated convergence are unclear. Exploring dynamic localization of APP/BACE-1 in cultured hippocampal neurons, we found that after synthesis via the secretory-pathway, dendritic APP/BACE-1-containing vesicles are largely segregated in physiologic states. While BACE-1 is largely sorted into acidic recycling endosomes, APP is conveyed in Golgi-derived vesicles. However upon activity-induction – a known trigger of the amyloidogenic pathway – APP is routed into BACE-1-positive recycling endosomes via a clathrin-dependent mechanism. A partitioning/convergence of APP/BACE-1 vesicles is also apparent in control/AD brains respectively. Considering BACE-1 is optimally active in an acidic environment, our experiments suggest that neurons have evolved trafficking strategies that normally limit APP/BACE-1 proximity; and also uncover a pathway routing APP into BACE-1-containing organelles – triggering amyloidogenesis. PMID:23931995

Regulation of the Hippo-YAP Pathway by Glucose Sensor O-GlcNAcylation.

PubMed

Peng, Changmin; Zhu, Yue; Zhang, Wanjun; Liao, Qinchao; Chen, Yali; Zhao, Xinyuan; Guo, Qiang; Shen, Pan; Zhen, Bei; Qian, Xiaohong; Yang, Dong; Zhang, Jin-San; Xiao, Dongguang; Qin, Weijie; Pei, Huadong

2017-11-02

The Hippo pathway is crucial in organ size control and tissue homeostasis, with deregulation leading to cancer. An extracellular nutrition signal, such as glucose, regulates the Hippo pathway activation. However, the mechanisms are still not clear. Here, we found that the Hippo pathway is directly regulated by the hexosamine biosynthesis pathway (HBP) in response to metabolic nutrients. Mechanistically, the core component of Hippo pathway (YAP) is O-GlcNAcylated by O-GlcNAc transferase (OGT) at serine 109. YAP O-GlcNAcylation disrupts its interaction with upstream kinase LATS1, prevents its phosphorylation, and activates its transcriptional activity. And this activation is not dependent on AMPK. We also identified OGT as a YAP-regulated gene that forms a feedback loop. Finally, we confirmed that glucose-induced YAP O-GlcNAcylation and activation promoted tumorigenesis. Together, our data establish a molecular mechanism and functional significance of the HBP in directly linking extracellular glucose signal to the Hippo-YAP pathway and tumorigenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Anisotropic Covalency Contributions to Superexchange Pathways in Type One Copper Active Sites

PubMed Central

2015-01-01

Type one (T1) Cu sites deliver electrons to catalytic Cu active sites: the mononuclear type two (T2) Cu site in nitrite reductases (NiRs) and the trinuclear Cu cluster in the multicopper oxidases (MCOs). The T1 Cu and the remote catalytic sites are connected via a Cys-His intramolecular electron-transfer (ET) bridge, which contains two potential ET pathways: P1 through the protein backbone and P2 through the H-bond between the Cys and the His. The high covalency of the T1 Cu–S(Cys) bond is shown here to activate the T1 Cu site for hole superexchange via occupied valence orbitals of the bridge. This covalency-activated electronic coupling (HDA) facilitates long-range ET through both pathways. These pathways can be selectively activated depending on the geometric and electronic structure of the T1 Cu site and thus the anisotropic covalency of the T1 Cu–S(Cys) bond. In NiRs, blue (π-type) T1 sites utilize P1 and green (σ-type) T1 sites utilize P2, with P2 being more efficient. Comparing the MCOs to NiRs, the second-sphere environment changes the conformation of the Cys-His pathway, which selectively activates HDA for superexchange by blue π sites for efficient turnover in catalysis. These studies show that a given protein bridge, here Cys-His, provides different superexchange pathways and electronic couplings depending on the anisotropic covalencies of the donor and acceptor metal sites. PMID:25310460

17β-Estradiol activates GPER- and ESR1-dependent pathways inducing apoptosis in GC-2 cells, a mouse spermatocyte-derived cell line.

PubMed

Chimento, Adele; Sirianni, Rosa; Casaburi, Ivan; Ruggiero, Carmen; Maggiolini, Marcello; Andò, Sebastiano; Pezzi, Vincenzo

2012-05-15

In mammals, spontaneous apoptosis is observed particularly in differentiating spermatogonia and in spermatocytes. 17β-Estradiol (E2) in primary rat pachytene spermatocytes (PS) binds estrogen receptor α (ESR1) and GPER to activate EGFR/ERK/c-Jun pathway leading to up regulation of proapoptotic factor bax. Aim of this study was to clarify the effector pathway(s) controlling spermatocytes apoptosis using as model GC-2 cells, an immortalized mouse pachytene spermatocyte-derived cell line, which reproduces primary cells responses to E2. In fact, in GC-2 cells we observed that ESR1 and GPER activation caused rapid ERK and c-Jun phosphorylation, bax up-regulation, events associated with apoptosis. We further investigated the apoptotic mechanism demonstrating that E2, as well as ESR1 and GPER specific agonists, induced sustained ERK, c-Jun and p38 phosphorylation, Cytochrome c release, caspase 3 and endogenous substrate Poly (ADP-ribose) polymerase (PARP) activation and increased expression of cell cycle inhibitor p21. When ESR1 or GPER expression was silenced, E2 was still able to decrease cell proliferation, only the concomitant silencing abolished E2 effect. These results indicate that GC-2 cells are a valid cell model to study E2-dependent apoptosis in spermatocytes and show that E2, activating both ESR1 and GPER, is able to induce an ERK1/2, c-Jun and p38-dependent mitochondrion apoptotic pathway in this cell type. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

The PI3K/Akt pathway is required for LPS activation of microglial cells.

PubMed

Saponaro, Concetta; Cianciulli, Antonia; Calvello, Rosa; Dragone, Teresa; Iacobazzi, Francesco; Panaro, Maria Antonietta

2012-10-01

Upregulation of inflammatory responses in the brain is associated with a number of neurodegenerative diseases. Microglia are activated in neurodegenerative diseases, producing pro-inflammatory mediators. Critically, lipopolysaccharide (LPS)-induced microglial activation causes dopaminergic neurodegeneration in vitro and in vivo. The signaling mechanisms triggered by LPS to stimulate the release of pro-inflammatory mediators in microglial cells are still incompletely understood. To further explore the mechanisms of LPS-mediated inflammatory response of microglial cells, we studied the role of phosphatidylinositol 3-kinase (PI3K)/Akt signal transduction pathways known to be activated by toll-like receptor-4 signaling through LPS. In the current study, we report that the activation profile of LPS-induced pAkt activation preceded those of LPS-induced NF-κB activation, suggesting a role for PI3K/Akt in the pathway activation of NF-κB-dependent inflammatory responses of activated microglia. These results, providing the first evidence that PI3K dependent signaling is involved in the inflammatory responses of microglial cells following LPS stimulation, may be useful in preventing inflammatory based neurodegenerative processes.

A cardiac pathway of cyclic GMP-independent signaling of guanylyl cyclase A, the receptor for atrial natriuretic peptide

PubMed Central

Klaiber, Michael; Dankworth, Beatrice; Kruse, Martin; Hartmann, Michael; Nikolaev, Viacheslav O.; Yang, Ruey-Bing; Völker, Katharina; Gaßner, Birgit; Oberwinkler, Heike; Feil, Robert; Freichel, Marc; Groschner, Klaus; Skryabin, Boris V.; Frantz, Stefan; Birnbaumer, Lutz; Pongs, Olaf; Kuhn, Michaela

2011-01-01

Cardiac atrial natriuretic peptide (ANP) regulates arterial blood pressure, moderates cardiomyocyte growth, and stimulates angiogenesis and metabolism. ANP binds to the transmembrane guanylyl cyclase (GC) receptor, GC-A, to exert its diverse functions. This process involves a cGMP-dependent signaling pathway preventing pathological [Ca2+]i increases in myocytes. In chronic cardiac hypertrophy, however, ANP levels are markedly increased and GC-A/cGMP responses to ANP are blunted due to receptor desensitization. Here we show that, in this situation, ANP binding to GC-A stimulates a unique cGMP-independent signaling pathway in cardiac myocytes, resulting in pathologically elevated intracellular Ca2+ levels. This pathway involves the activation of Ca2+‐permeable transient receptor potential canonical 3/6 (TRPC3/C6) cation channels by GC-A, which forms a stable complex with TRPC3/C6 channels. Our results indicate that the resulting cation influx activates voltage-dependent L-type Ca2+ channels and ultimately increases myocyte Ca2+i levels. These observations reveal a dual role of the ANP/GC-A–signaling pathway in the regulation of cardiac myocyte Ca2+i homeostasis. Under physiological conditions, activation of a cGMP-dependent pathway moderates the Ca2+i-enhancing action of hypertrophic factors such as angiotensin II. By contrast, a cGMP-independent pathway predominates under pathophysiological conditions when GC-A is desensitized by high ANP levels. The concomitant rise in [Ca2+]i might increase the propensity to cardiac hypertrophy and arrhythmias. PMID:22027011

Hydrogen oxidation mechanisms on Ni/yttria stabilized zirconia anodes: Separation of reaction pathways by geometry variation of pattern electrodes

NASA Astrophysics Data System (ADS)

Doppler, M. C.; Fleig, J.; Bram, M.; Opitz, A. K.

2018-03-01

Nickel/yttria stabilized zirconia (YSZ) electrodes are affecting the overall performance of solid oxide fuel cells (SOFCs) in general and strongly contribute to the cell resistance in case of novel metal supported SOFCs in particular. The electrochemical fuel conversion mechanisms in these electrodes are, however, still only partly understood. In this study, micro-structured Ni thin film electrodes on YSZ with 15 different geometries are utilized to investigate reaction pathways for the hydrogen electro-oxidation at Ni/YSZ anodes. From electrodes with constant area but varying triple phase boundary (TPB) length a contribution to the electro-catalytic activity is found that does not depend on the TPB length. This additional activity could clearly be attributed to a yet unknown reaction pathway scaling with the electrode area. It is shown that this area related pathway has significantly different electrochemical behavior compared to the TPB pathway regarding its thermal activation, sulfur poisoning behavior, and H2/H2O partial pressure dependence. Moreover, possible reaction mechanisms of this reaction pathway are discussed, identifying either a pathway based on hydrogen diffusion through Ni with water release at the TPB or a path with oxygen diffusion through Ni to be a very likely explanation for the experimental results.

Refining Pathways: A Model Comparison Approach

PubMed Central

Moffa, Giusi; Erdmann, Gerrit; Voloshanenko, Oksana; Hundsrucker, Christian; Sadeh, Mohammad J.; Boutros, Michael; Spang, Rainer

2016-01-01

Cellular signalling pathways consolidate multiple molecular interactions into working models of signal propagation, amplification, and modulation. They are described and visualized as networks. Adjusting network topologies to experimental data is a key goal of systems biology. While network reconstruction algorithms like nested effects models are well established tools of computational biology, their data requirements can be prohibitive for their practical use. In this paper we suggest focussing on well defined aspects of a pathway and develop the computational tools to do so. We adapt the framework of nested effect models to focus on a specific aspect of activated Wnt signalling in HCT116 colon cancer cells: Does the activation of Wnt target genes depend on the secretion of Wnt ligands or do mutations in the signalling molecule β-catenin make this activation independent from them? We framed this question into two competing classes of models: Models that depend on Wnt ligands secretion versus those that do not. The model classes translate into restrictions of the pathways in the network topology. Wnt dependent models are more flexible than Wnt independent models. Bayes factors are the standard Bayesian tool to compare different models fairly on the data evidence. In our analysis, the Bayes factors depend on the number of potential Wnt signalling target genes included in the models. Stability analysis with respect to this number showed that the data strongly favours Wnt ligands dependent models for all realistic numbers of target genes. PMID:27248690

Interaction of AIP with protein kinase A (cAMP-dependent protein kinase).

PubMed

Schernthaner-Reiter, Marie Helene; Trivellin, Giampaolo; Stratakis, Constantine A

2018-05-02

Germline mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene cause mostly somatotropinomas and/or prolactinomas in a subset of familial isolated pituitary adenomas (FIPA). AIP has been shown to interact with phosphodiesterases (PDEs) and G proteins, suggesting a link to the cyclic AMP (cAMP)-dependent protein kinase (PKA) pathway. Upregulation of PKA is seen in sporadic somatotropinomas that carry GNAS1 mutations, and those in Carney complex that are due to PRKAR1A mutations. To elucidate the mechanism of AIP-dependent pituitary tumorigenesis, we studied potential functional and physical interactions of AIP with PKA's main subunits PRKAR1A (R1α) and PRKACA (Cα). We found that AIP physically interacts with both R1α and Cα; this interaction is enhanced when all three components are present, but maintained during Cα-R1α dissociation by PKA pathway activation, indicating that AIP binds Cα/R1α both in complex and separately. The interaction between AIP and R1α/Cα is reduced when the frequent AIP pathogenic mutation p.R304* is present. AIP protein levels are regulated both by translation and the ubiquitin/proteasome pathway and Cα stabilizes both AIP and R1α protein levels. AIP reduction by siRNA leads to an increase of PKA pathway activity, which is disproportionately enhanced during PDE4-inhibition. We show that AIP interacts with the PKA pathway on multiple levels, including a physical interaction with both the main regulatory (R1α) and catalytic (Cα) PKA subunits and a functional interaction with PDE4-dependent PKA activation. These findings provide novel insights on the mechanisms of AIP-dependent pituitary tumorigenesis.

FasL-triggered death of Jurkat cells requires caspase 8-induced, ATP-dependent cross-talk between Fas and the purinergic receptor P2X(7).

PubMed

Aguirre, Adam; Shoji, Kenji F; Sáez, Juan C; Henríquez, Mauricio; Quest, Andrew F G

2013-02-01

Fas ligation via the ligand FasL activates the caspase-8/caspase-3-dependent extrinsic death pathway. In so-called type II cells, an additional mechanism involving tBid-mediated caspase-9 activation is required to efficiently trigger cell death. Other pathways linking FasL-Fas interaction to activation of the intrinsic cell death pathway remain unknown. However, ATP release and subsequent activation of purinergic P2X(7) receptors (P2X(7)Rs) favors cell death in some cells. Here, we evaluated the possibility that ATP release downstream of caspase-8 via pannexin1 hemichannels (Panx1 HCs) and subsequent activation of P2X(7)Rs participate in FasL-stimulated cell death. Indeed, upon FasL stimulation, ATP was released from Jurkat cells in a time- and caspase-8-dependent manner. Fas and Panx1 HCs colocalized and inhibition of the latter, but not connexin hemichannels, reduced FasL-induced ATP release. Extracellular apyrase, which hydrolyzes ATP, reduced FasL-induced death. Also, oxidized-ATP or Brilliant Blue G, two P2X(7)R blockers, reduced FasL-induced caspase-9 activation and cell death. These results represent the first evidence indicating that the two death receptors, Fas and P2X(7)R connect functionally via caspase-8 and Panx1 HC-mediated ATP release to promote caspase-9/caspase-3-dependent cell death in lymphoid cells. Thus, a hitherto unsuspected route was uncovered connecting the extrinsic to the intrinsic pathway to amplify death signals emanating from the Fas receptor in type II cells. Copyright © 2012 Wiley Periodicals, Inc.

Cynaropicrin attenuates UVB-induced oxidative stress via the AhR-Nrf2-Nqo1 pathway.

PubMed

Takei, Kenjiro; Hashimoto-Hachiya, Akiko; Takahara, Masakazu; Tsuji, Gaku; Nakahara, Takeshi; Furue, Masutaka

2015-04-16

Due to its antioxidant and anti-inflammatory activities, artichoke (Cynara scolymus) has been used as folk medicine to treat various diseases. Cynaropicrin (Cyn), a sesquiterpene lactone, is the major bioactive phytochemical in the artichoke; however, its pharmacological mechanism remains unknown. Because some phytochemicals exert their antioxidant activity by activating aryl hydrocarbon receptor (AhR), leading to subsequent induction of the antioxidant pathway including nuclear factor E2-related factor 2 (Nrf2) and quinone oxidoreductase 1 (Nqo1), we investigated whether Cyn also activates the AhR-Nrf2-Nqo1 pathway. Cyn indeed induced the activation (nuclear translocation) of AhR, leading to nuclear translocation of Nrf2 and dose-dependent upregulation of Nrf2 and Nqo1 mRNAs in human keratinocytes. The Cyn-induced AhR-Nrf2-Nqo1 activation was AhR- and Nrf2-dependent, as demonstrated by the observation that it was absent in keratinocytes transfected by siRNA against either AhR or Nrf2. In accordance with these findings, Cyn actively inhibited generation of reactive oxygen species from keratinocytes irradiated with ultraviolet B (UVB) in a Nrf2-dependent manner. Cyn also inhibited the production of proinflammatory cytokines such as interleukin 6 and tumor necrosis factor-α from UVB-treated keratinocytes. Our findings demonstrate that Cyn is a potent activator of the AhR-Nrf2-Nqo1 pathway, and could therefore be applied to prevention of UVB-induced photo aging. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Cilostazol improves high glucose-induced impaired angiogenesis in human endothelial progenitor cells and vascular endothelial cells as well as enhances vasculoangiogenesis in hyperglycemic mice mediated by the adenosine monophosphate-activated protein kinase pathway.

PubMed

Tseng, Shih-Ya; Chao, Ting-Hsing; Li, Yi-Heng; Liu, Ping-Yen; Lee, Cheng-Han; Cho, Chung-Lung; Wu, Hua-Lin; Chen, Jyh-Hong

2016-04-01

Cilostazol is an antiplatelet agent with vasodilatory effects that works by increasing intracellular concentrations of cyclic adenosine monophosphate (cAMP). This study investigated the effects of cilostazol in preventing high glucose (HG)-induced impaired angiogenesis and examined the potential mechanisms involving activation of AMP-activated protein kinase (AMPK). Assays for colony formation, adhesion, proliferation, migration, and vascular tube formation were used to determine the effect of cilostazol in HG-treated endothelial progenitor cells (EPCs) or human umbilical vein endothelial cells (HUVECs). Animal-based assays were performed in hyperglycemic ICR mice undergoing hind limb ischemia. An immnunoblotting assay was used to identify the expression and activation of signaling molecules in vitro and in vivo. Cilostazol treatment significantly restored endothelial function in EPCs and HUVECs through activation of AMPK/acetyl-coenzyme A carboxylase (ACC)-dependent pathways and cAMP/protein kinase A (PKA)-dependent pathways. Recovery of blood flow in the ischemic hind limb and the population of circulating CD34(+) cells were significantly improved in cilostazol-treated mice, and these effects were abolished by local AMPK knockdown. Cilostazol increased the phosphorylation of AMPK/ACC and Akt/endothelial nitric oxide synthase signaling molecules in parallel with or downstream of the cAMP/PKA-dependent signaling pathway in vitro and in vivo. Cilostazol prevents HG-induced endothelial dysfunction in EPCs and HUVECs and enhances angiogenesis in hyperglycemic mice by interactions with a broad signaling network, including activation of AMPK/ACC and probably cAMP/PKA pathways. Copyright © 2016 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.

Identification of a Novel Gnao-Mediated Alternate Olfactory Signaling Pathway in Murine OSNs.

PubMed

Scholz, Paul; Mohrhardt, Julia; Jansen, Fabian; Kalbe, Benjamin; Haering, Claudia; Klasen, Katharina; Hatt, Hanns; Osterloh, Sabrina

2016-01-01

It is generally agreed that in olfactory sensory neurons (OSNs), the binding of odorant molecules to their specific olfactory receptor (OR) triggers a cAMP-dependent signaling cascade, activating cyclic-nucleotide gated (CNG) channels. However, considerable controversy dating back more than 20 years has surrounded the question of whether alternate signaling plays a role in mammalian olfactory transduction. In this study, we demonstrate a specific alternate signaling pathway in Olfr73-expressing OSNs. Methylisoeugenol (MIEG) and at least one other known weak Olfr73 agonist (Raspberry Ketone) trigger a signaling cascade independent from the canonical pathway, leading to the depolarization of the cell. Interestingly, this pathway is mediated by Gnao activation, leading to Cl(-) efflux; however, the activation of adenylyl cyclase III (ACIII), the recruitment of Ca(2+) from extra-or intracellular stores, and phosphatidylinositol 3-kinase-dependent signaling (PI signaling) are not involved. Furthermore, we demonstrated that our newly identified pathway coexists with the canonical olfactory cAMP pathway in the same OSN and can be triggered by the same OR in a ligand-selective manner. We suggest that this pathway might reflect a mechanism for odor recognition predominantly used in early developmental stages before olfactory cAMP signaling is fully developed. Taken together, our findings support the existence of at least one odor-induced alternate signal transduction pathway in native OSNs mediated by Olfr73 in a ligand-selective manner.

CD40-TNF activation in mice induces extended sickness behavior syndrome co-incident with but not dependent on activation of the kynurenine pathway.

PubMed

Cathomas, Flurin; Fuertig, Rene; Sigrist, Hannes; Newman, Gregory N; Hoop, Vanessa; Bizzozzero, Manuela; Mueller, Andreas; Luippold, Andreas; Ceci, Angelo; Hengerer, Bastian; Seifritz, Erich; Fontana, Adriano; Pryce, Christopher R

2015-11-01

The similarity between sickness behavior syndrome (SBS) in infection and autoimmune disorders and certain symptoms in major depressive disorder (MDD), and the high co-morbidity of autoimmune disorders and MDD, constitutes some of the major evidence for the immune-inflammation hypothesis of MDD. CD40 ligand-CD40 immune-activation is important in host response to infection and in development of autoimmunity. Mice given a single intra-peritoneal injection of CD40 agonist antibody (CD40AB) develop SBS for 2-3days characterized by weight loss and increased sleep, effects that are dependent on the cytokine, tumor necrosis factor (TNF). Here we report that CD40AB also induces behavioral effects that extend beyond acute SBS and co-occur with but are not mediated by kynurenine pathway activation and recovery. CD40AB led to decreased saccharin drinking (days 1-7) and decreased Pavlovian fear conditioning (days 5-6), and was without effect on physical fatigue (day 5). These behavioral effects co-occurred with increased plasma and brain levels of kynurenine and its metabolites (days 1-7/8). Co-injection of TNF blocker etanercept with CD40AB prevented each of SBS, reduced saccharin drinking, and kynurenine pathway activation in plasma and brain. Repeated oral administration of a selective indoleamine 2,3-dioxygenase (IDO) inhibitor blocked activation of the kynurenine pathway but was without effect on SBS and saccharin drinking. This study provides novel evidence that CD40-TNF activation induces deficits in saccharin drinking and Pavlovian fear learning and activates the kynurenine pathway, and that CD40-TNF activation of the kynurenine pathway is not necessary for induction of the acute or extended SBS effects. Copyright © 2015 Elsevier Inc. All rights reserved.

Beta-Adrenergic Receptor Activation during Distinct Patterns of Stimulation Critically Modulates the PKA-Dependence of LTP in the Mouse Hippocampus

ERIC Educational Resources Information Center

Gelinas, Jennifer N.; Tenorio, Gustavo; Lemon, Neal; Abel, Ted; Nguyen, Peter V.

2008-01-01

Activation of Beta-adrenergic receptors (Beta-ARs) enhances hippocampal memory consolidation and long-term potentiation (LTP), a likely mechanism for memory storage. One signaling pathway linked to Beta-AR activation is the cAMP-PKA pathway. PKA is critical for the consolidation of hippocampal long-term memory and for the expression of some forms…

Requirement for pectin methyl esterase and preference for fragmented over native pectins for wall-associated kinase-activated, EDS1/PAD4-dependent stress response in Arabidopsis.

PubMed

Kohorn, Bruce D; Kohorn, Susan L; Saba, Nicholas J; Martinez, Victoriano Meco

2014-07-04

The wall-associated kinases (WAKs) have a cytoplasmic protein kinase domain that spans the plasma membrane and binds pectin in the extracellular matrix of plants. WAKs are required for cell expansion during Arabidopsis seedling development but are also an integral part of the response to pathogens and stress that present oligogalacturonides (OGs), which subsequently bind to WAKs and activate a MPK6 (mitogen-activated protein kinase)-dependent pathway. It was unclear how WAKs distinguish native pectin polymers and OGs to activate one or the other of these two pathways. A dominant allele of WAK2 constitutively activates the stress response, and we show here that the effect is dependent upon EDS1 and PAD4, transcriptional activators involved in the pathogen response. Moreover, the WAK2 dominant allele is suppressed by a null allele of a pectin methyl esterase (PME3) whose activity normally leads to cross-linking of pectins in the cell wall. Although OGs activate a transcriptional response in wild type, the response is enhanced in a pme3/pme3 null, consistent with a competition by OG and native polymers for activation of WAKs. This provides a plausible mechanism for WAKs to distinguish an expansion from a stress pathway. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Shape-Dependent Skin Penetration of Silver Nanoparticles: Does It Really Matter?

PubMed Central

Tak, Yu Kyung; Pal, Sukdeb; Naoghare, Pravin K.; Rangasamy, Sabarinathan; Song, Joon Myong

2015-01-01

Advancements in nano-structured materials have facilitated several applications of nanoparticles (NPs). Skin penetration of NPs is a crucial factor for designing suitable topical antibacterial agents with low systemic toxicity. Available reports focus on size-dependent skin penetration of NPs, mainly through follicular pathways. Herein, for the first time, we demonstrate a proof-of-concept study that entails variations in skin permeability and diffusion coefficients, penetration rates and depth-of-penetration of differently shaped silver NPs (AgNPs) via intercellular pathways using both in vitro and in vivo models. The antimicrobial activity of AgNPs is known. Different shapes of AgNPs may exhibit diverse antimicrobial activities and skin penetration capabilities depending upon their active metallic facets. Consideration of the shape dependency of AgNPs in antimicrobial formulations could help developing an ideal topical agent with the highest efficacy and low systemic toxicity. PMID:26584777

The DEG/ENaC cation channel protein UNC-8 drives activity-dependent synapse removal in remodeling GABAergic neurons

PubMed Central

Miller-Fleming, Tyne W; Petersen, Sarah C; Manning, Laura; Matthewman, Cristina; Gornet, Megan; Beers, Allison; Hori, Sayaka; Mitani, Shohei; Bianchi, Laura; Richmond, Janet; Miller, David M

2016-01-01

Genetic programming and neural activity drive synaptic remodeling in developing neural circuits, but the molecular components that link these pathways are poorly understood. Here we show that the C. elegans Degenerin/Epithelial Sodium Channel (DEG/ENaC) protein, UNC-8, is transcriptionally controlled to function as a trigger in an activity-dependent mechanism that removes synapses in remodeling GABAergic neurons. UNC-8 cation channel activity promotes disassembly of presynaptic domains in DD type GABA neurons, but not in VD class GABA neurons where unc-8 expression is blocked by the COUP/TF transcription factor, UNC-55. We propose that the depolarizing effect of UNC-8-dependent sodium import elevates intracellular calcium in a positive feedback loop involving the voltage-gated calcium channel UNC-2 and the calcium-activated phosphatase TAX-6/calcineurin to initiate a caspase-dependent mechanism that disassembles the presynaptic apparatus. Thus, UNC-8 serves as a link between genetic and activity-dependent pathways that function together to promote the elimination of GABA synapses in remodeling neurons. DOI: http://dx.doi.org/10.7554/eLife.14599.001 PMID:27403890

Genetic Feedback Regulation of Frontal Cortical Neuronal Ensembles Through Activity-Dependent Arc Expression and Dopaminergic Input.

PubMed

Mastwal, Surjeet; Cao, Vania; Wang, Kuan Hong

2016-01-01

Mental functions involve coordinated activities of specific neuronal ensembles that are embedded in complex brain circuits. Aberrant neuronal ensemble dynamics is thought to form the neurobiological basis of mental disorders. A major challenge in mental health research is to identify these cellular ensembles and determine what molecular mechanisms constrain their emergence and consolidation during development and learning. Here, we provide a perspective based on recent studies that use activity-dependent gene Arc/Arg3.1 as a cellular marker to identify neuronal ensembles and a molecular probe to modulate circuit functions. These studies have demonstrated that the transcription of Arc is activated in selective groups of frontal cortical neurons in response to specific behavioral tasks. Arc expression regulates the persistent firing of individual neurons and predicts the consolidation of neuronal ensembles during repeated learning. Therefore, the Arc pathway represents a prototypical example of activity-dependent genetic feedback regulation of neuronal ensembles. The activation of this pathway in the frontal cortex starts during early postnatal development and requires dopaminergic (DA) input. Conversely, genetic disruption of Arc leads to a hypoactive mesofrontal dopamine circuit and its related cognitive deficit. This mutual interaction suggests an auto-regulatory mechanism to amplify the impact of neuromodulators and activity-regulated genes during postnatal development. Such a mechanism may contribute to the association of mutations in dopamine and Arc pathways with neurodevelopmental psychiatric disorders. As the mesofrontal dopamine circuit shows extensive activity-dependent developmental plasticity, activity-guided modulation of DA projections or Arc ensembles during development may help to repair circuit deficits related to neuropsychiatric disorders.

Real-time cell toxicity profiling of Tox21 10K compounds reveals cytotoxicity dependent toxicity pathway linkage

PubMed Central

Huang, Ruili; Lin, Ja-An; Sedykh, Alexander; Zhao, Jinghua; Tice, Raymond R.; Paules, Richard S.; Xia, Menghang; Auerbach, Scott S.

2017-01-01

Cytotoxicity is a commonly used in vitro endpoint for evaluating chemical toxicity. In support of the U.S. Tox21 screening program, the cytotoxicity of ~10K chemicals was interrogated at 0, 8, 16, 24, 32, & 40 hours of exposure in a concentration dependent fashion in two cell lines (HEK293, HepG2) using two multiplexed, real-time assay technologies. One technology measures the metabolic activity of cells (i.e., cell viability, glo) while the other evaluates cell membrane integrity (i.e., cell death, flor). Using glo technology, more actives and greater temporal variations were seen in HEK293 cells, while results for the flor technology were more similar across the two cell types. Chemicals were grouped into classes based on their cytotoxicity kinetics profiles and these classes were evaluated for their associations with activity in the Tox21 nuclear receptor and stress response pathway assays. Some pathways, such as the activation of H2AX, were associated with the fast-responding cytotoxicity classes, while others, such as activation of TP53, were associated with the slow-responding cytotoxicity classes. By clustering pathways based on their degree of association to the different cytotoxicity kinetics labels, we identified clusters of pathways where active chemicals presented similar kinetics of cytotoxicity. Such linkages could be due to shared underlying biological processes between pathways, for example, activation of H2AX and heat shock factor. Others involving nuclear receptor activity are likely due to shared chemical structures rather than pathway level interactions. Based on the linkage between androgen receptor antagonism and Nrf2 activity, we surmise that a subclass of androgen receptor antagonists cause cytotoxicity via oxidative stress that is associated with Nrf2 activation. In summary, the real-time cytotoxicity screen provides informative chemical cytotoxicity kinetics data related to their cytotoxicity mechanisms, and with our analysis, it is possible to formulate mechanism-based hypotheses on the cytotoxic properties of the tested chemicals. PMID:28531190

The cAMP Response Element Binding protein (CREB) is activated by Insulin-like Growth Factor-1 (IGF-1) and regulates myostatin gene expression in skeletal myoblast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zuloaga, R.; Fuentes, E.N.; Molina, A.

2013-10-18

Highlights: •IGF-1 induces the activation of CREB via IGF-1R/PI3K/PLC signaling pathway. •Calcium dependent signaling pathways regulate myostatin gene expression. •IGF-1 regulates myostatin gene expression via CREB transcription in skeletal myoblast. -- Abstract: Myostatin, a member of the Transforming Growth Factor beta (TGF-β) superfamily, plays an important role as a negative regulator of skeletal muscle growth and differentiation. We have previously reported that IGF-1 induces a transient myostatin mRNA expression, through the activation of the Nuclear Factor of Activated T cells (NFAT) in an IP{sub 3}/calcium-dependent manner. Here we examined the activation of CREB transcription factor as downstream targets of IGF-1more » during myoblast differentiation and its role as a regulator of myostatin gene expression. In cultured skeletal myoblast, IGF-1 induced the phosphorylation and transcriptional activation of CREB via IGF-1 Receptor/Phosphatidylinositol 3-Kinase (PI3K)/Phospholipase C gamma (PLC γ), signaling pathways. Also, IGF-1 induced calcium-dependent molecules such as Calmodulin Kinase II (CaMK II), Extracellular signal-regulated Kinases (ERK), Protein Kinase C (PKC). Additionally, we examined myostatin mRNA levels and myostatin promoter activity in differentiated myoblasts stimulated with IGF-1. We found a significant increase in mRNA contents of myostatin and its reporter activity after treatment with IGF-1. The expression of myostatin in differentiated myoblast was downregulated by the transfection of siRNA–CREB and by pharmacological inhibitors of the signaling pathways involved in CREB activation. By using pharmacological and genetic approaches together these data demonstrate that IGF-1 regulates the myostatin gene expression via CREB transcription factor during muscle cell differentiation.« less

Akt/PKB activation and insulin signaling: a novel insulin signaling pathway in the treatment of type 2 diabetes.

PubMed

Mackenzie, Richard Wa; Elliott, Bradley T

2014-01-01

Type 2 diabetes is a metabolic disease categorized primarily by reduced insulin sensitivity, β-cell dysfunction, and elevated hepatic glucose production. Treatments reducing hyperglycemia and the secondary complications that result from these dysfunctions are being sought after. Two distinct pathways encourage glucose transport activity in skeletal muscle, ie, the contraction-stimulated pathway reliant on Ca(2+)/5'-monophosphate-activated protein kinase (AMPK)-dependent mechanisms and an insulin-dependent pathway activated via upregulation of serine/threonine protein kinase Akt/PKB. Metformin is an established treatment for type 2 diabetes due to its ability to increase peripheral glucose uptake while reducing hepatic glucose production in an AMPK-dependent manner. Peripheral insulin action is reduced in type 2 diabetics whereas AMPK signaling remains largely intact. This paper firstly reviews AMPK and its role in glucose uptake and then focuses on a novel mechanism known to operate via an insulin-dependent pathway. Inositol hexakisphosphate (IP6) kinase 1 (IP6K1) produces a pyrophosphate group at the position of IP6 to generate a further inositol pyrophosphate, ie, diphosphoinositol pentakisphosphate (IP7). IP7 binds with Akt/PKB at its pleckstrin homology domain, preventing interaction with phosphatidylinositol 3,4,5-trisphosphate, and therefore reducing Akt/PKB membrane translocation and insulin-stimulated glucose uptake. Novel evidence suggesting a reduction in IP7 production via IP6K1 inhibition represents an exciting therapeutic avenue in the treatment of insulin resistance. Metformin-induced activation of AMPK is a key current intervention in the management of type 2 diabetes. However, this treatment does not seem to improve peripheral insulin resistance. In light of this evidence, we suggest that inhibition of IP6K1 may increase insulin sensitivity and provide a novel research direction in the treatment of insulin resistance.

Berberine regulates neurite outgrowth through AMPK-dependent pathways by lowering energy status

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, Jiaqi; Cao, Yuanzhao; Cheng, Kuoyuan

2015-06-10

As a widely used anti-bacterial agent and a metabolic inhibitor as well as AMP-activated protein kinase (AMPK) activator, berberine (BBR) has been shown to cross the blood–brain barrier. Its efficacy has been investigated in various disease models of the central nervous system. Neurite outgrowth is critical for nervous system development and is a highly energy-dependent process regulated by AMPK-related pathways. In the present study, we aimed to investigate the effects of BBR on AMPK activation and neurite outgrowth in neurons. The neurite outgrowth of primary rat cortical neurons at different stages of polarization was monitored after exposure of BBR. Intracellularmore » energy level, AMPK activation and polarity-related pathways were also inspected. The results showed that BBR suppressed neurite outgrowth and affected cytoskeleton stability in the early stages of neuronal polarization, which was mediated by lowered energy status and AMPK activation. Liver kinase B1 and PI3K–Akt–GSK3β signaling pathways were also involved. In addition, mitochondrial dysfunction and endoplasmic reticulum stress contributed to the lowered energy status induced by BBR. This study highlighted the knowledge of the complex activities of BBR in neurons and corroborated the significance of energy status during the neuronal polarization. - Highlights: • BBR inhibited neurite outgrowth in early stages of neuronal development. • Lowered neuronal energy status was induced by BBR treatment. • Neuronal energy stress induced by BBR activated AMPK-related pathways. • BBR induced mitochondrial dysfunction and endoplasmic reticulum stress.« less

Synaptic activity regulates AMPA receptor trafficking through different recycling pathways

PubMed Central

Zheng, Ning; Jeyifous, Okunola; Munro, Charlotte; Montgomery, Johanna M; Green, William N

2015-01-01

Changes in glutamatergic synaptic strength in brain are dependent on AMPA-type glutamate receptor (AMPAR) recycling, which is assumed to occur through a single local pathway. In this study, we present evidence that AMPAR recycling occurs through different pathways regulated by synaptic activity. Without synaptic stimulation, most AMPARs recycled in dynamin-independent endosomes containing the GTPase, Arf6. Few AMPARs recycled in dynamin-dependent endosomes labeled by transferrin receptors (TfRs). AMPAR recycling was blocked by alterations in the GTPase, TC10, which co-localized with Arf6 endosomes. TC10 mutants that reduced AMPAR recycling had no effect on increased AMPAR levels with long-term potentiation (LTP) and little effect on decreased AMPAR levels with long-term depression. However, internalized AMPAR levels in TfR-containing recycling endosomes increased after LTP, indicating increased AMPAR recycling through the dynamin-dependent pathway with synaptic plasticity. LTP-induced AMPAR endocytosis is inconsistent with local recycling as a source of increased surface receptors, suggesting AMPARs are trafficked from other sites. DOI: http://dx.doi.org/10.7554/eLife.06878.001 PMID:25970033

β-Adrenergic induced SR Ca2+ leak is mediated by an Epac-NOS pathway.

PubMed

Pereira, Laëtitia; Bare, Dan J; Galice, Samuel; Shannon, Thomas R; Bers, Donald M

2017-07-01

Cardiac β-adrenergic receptors (β-AR) and Ca 2+ -Calmodulin dependent protein kinase (CaMKII) regulate both physiological and pathophysiological Ca 2+ signaling. Elevated diastolic Ca 2+ leak from the sarcoplasmic reticulum (SR) contributes to contractile dysfunction in heart failure and to arrhythmogenesis. β-AR activation is known to increase SR Ca 2+ leak via CaMKII-dependent phosphorylation of the ryanodine receptor. Two independent and reportedly parallel pathways have been implicated in this β-AR-CaMKII cascade, one involving exchange protein directly activated by cAMP (Epac2) and another involving nitric oxide synthase 1 (NOS1). Here we tested whether Epac and NOS function in a single series pathway to increase β-AR induced and CaMKII-dependent SR Ca 2+ leak. Leak was measured as both Ca 2+ spark frequency and tetracaine-induced shifts in SR Ca 2+ , in mouse and rabbit ventricular myocytes. Direct Epac activation by 8-CPT (8-(4-chlorophenylthio)-2'-O-methyl-cAMP) mimicked β-AR-induced SR Ca 2+ leak, and both were blocked by NOS inhibition. The same was true for myocyte CaMKII activation (assessed via a FRET-based reporter) and ryanodine receptor phosphorylation. Inhibitor and phosphorylation studies also implicated phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt) downstream of Epac and above NOS activation in this pathway. We conclude that these two independently characterized parallel pathways function mainly via a single series arrangement (β-AR-cAMP-Epac-PI3K-Akt-NOS1-CaMKII) to mediate increased SR Ca 2+ leak. Thus, for β-AR activation the cAMP-PKA branch effects inotropy and lusitropy (by effects on Ca 2+ current and SR Ca 2+ -ATPase), this cAMP-Epac-NOS pathway increases pathological diastolic SR Ca 2+ leak. This pathway distinction may allow novel SR Ca 2+ leak therapeutic targeting in treatment of arrhythmias in heart failure that spare the inotropic and lusitropic effects of the PKA branch. Copyright © 2017 Elsevier Ltd. All rights reserved.

Caspase-3 dependent nitrergic neuronal apoptosis following cavernous nerve injury is mediated via RhoA and ROCK activation in major pelvic ganglion.

PubMed

Hannan, Johanna L; Matsui, Hotaka; Sopko, Nikolai A; Liu, Xiaopu; Weyne, Emmanuel; Albersen, Maarten; Watson, Joseph W; Hoke, Ahmet; Burnett, Arthur L; Bivalacqua, Trinity J

2016-07-08

Axonal injury due to prostatectomy leads to Wallerian degeneration of the cavernous nerve (CN) and erectile dysfunction (ED). Return of potency is dependent on axonal regeneration and reinnervation of the penis. Following CN injury (CNI), RhoA and Rho-associated protein kinase (ROCK) increase in penile endothelial and smooth muscle cells. Previous studies indicate that nerve regeneration is hampered by activation of RhoA/ROCK pathway. We evaluated the role of RhoA/ROCK pathway in CN regulation following CNI using a validated rat model. CNI upregulated gene and protein expression of RhoA/ROCK and caspase-3 mediated apoptosis in the major pelvic ganglion (MPG). ROCK inhibitor (ROCK-I) prevented upregulation of RhoA/ROCK pathway as well as activation of caspase-3 in the MPG. Following CNI, there was decrease in the dimer to monomer ratio of neuronal nitric oxide synthase (nNOS) protein and lowered NOS activity in the MPG, which were prevented by ROCK-I. CNI lowered intracavernous pressure and impaired non-adrenergic non-cholinergic-mediated relaxation in the penis, consistent with ED. ROCK-I maintained the intracavernous pressure and non-adrenergic non-cholinergic-mediated relaxation in the penis following CNI. These results suggest that activation of RhoA/ROCK pathway mediates caspase-3 dependent apoptosis of nitrergic neurons in the MPG following CNI and that ROCK-I can prevent post-prostatectomy ED.

Calcineurin inhibitors recruit protein kinases JAK2 and JNK, TLR signaling and the UPR to activate NF-κB-mediated inflammatory responses in kidney tubular cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

González-Guerrero, Cristian, E-mail: cristian.gonzalez@fjd.es; Ocaña-Salceda, Carlos, E-mail: carlos.ocana@fjd.es; Berzal, Sergio, E-mail: sberzal@fjd.es

The calcineurin inhibitors (CNIs) cyclosporine (CsA) and tacrolimus are key drugs in current immunosuppressive regimes for solid organ transplantation. However, they are nephrotoxic and promote death and profibrotic responses in tubular cells. Moreover, renal inflammation is observed in CNI nephrotoxicity but the mechanisms are poorly understood. We have now studied molecular pathways leading to inflammation elicited by the CNIs in cultured and kidney tubular cells. Both CsA and tacrolimus elicited a proinflammatory response in tubular cells as evidenced by a transcriptomics approach. Transcriptomics also suggested several potential pathways leading to expression of proinflammatory genes. Validation and functional studies disclosed thatmore » in tubular cells, CNIs activated protein kinases such as the JAK2/STAT3 and TAK1/JNK/AP-1 pathways, TLR4/Myd88/IRAK signaling and the Unfolded Protein Response (UPR) to promote NF-κB activation and proinflammatory gene expression. CNIs also activated an Nrf2/HO-1-dependent compensatory response and the Nrf2 activator sulforaphane inhibited JAK2 and JNK activation and inflammation. A murine model of CsA nephrotoxicity corroborated activation of the proinflammatory pathways identified in cell cultures. Human CNIs nephrotoxicity was also associated with NF-κB, STAT3 and IRE1α activation. In conclusion, CNIs recruit several intracellular pathways leading to previously non-described proinflammatory actions in renal tubular cells. Identification of these pathways provides novel clues for therapeutic intervention to limit CNIs nephrotoxicity. - Highlights: • Molecular mechanisms modulating CNI renal inflammation were investigated. • Kinases, immune receptors and ER stress mediate the inflammatory response to CNIs. • Several intracellular pathways activate NF-κB in CNIs-treated tubular cells. • A NF-κB-dependent cytokine profile characterizes CNIs-induced inflammation. • CNI nephrotoxicity was associated to inflammatory events in mice and human.« less

Calcium/calmodulin-dependent protein kinase II activity regulates the proliferative potential of growth plate chondrocytes.

PubMed

Li, Yuwei; Ahrens, Molly J; Wu, Amy; Liu, Jennifer; Dudley, Andrew T

2011-01-01

For tissues that develop throughout embryogenesis and into postnatal life, the generation of differentiated cells to promote tissue growth is at odds with the requirement to maintain the stem cell/progenitor cell population to preserve future growth potential. In the growth plate cartilage, this balance is achieved in part by establishing a proliferative phase that amplifies the number of progenitor cells prior to terminal differentiation into hypertrophic chondrocytes. Here, we show that endogenous calcium/calmodulin-dependent protein kinase II (CamkII, also known as Camk2) activity is upregulated prior to hypertrophy and that loss of CamkII function substantially blocks the transition from proliferation to hypertrophy. Wnt signaling and Pthrp-induced phosphatase activity negatively regulate CamkII activity. Release of this repression results in activation of multiple effector pathways, including Runx2- and β-catenin-dependent pathways. We present an integrated model for the regulation of proliferation potential by CamkII activity that has important implications for studies of growth control and adult progenitor/stem cell populations.

Effects of low dose treatment of tributyltin on the regulation of estrogen receptor functions in MCF-7 cells.

PubMed

Sharan, Shruti; Nikhil, Kumar; Roy, Partha

2013-06-01

Endocrine disrupting chemicals are the natural/synthetic compounds which mimic or inhibit the actions of endogenous hormones. Organotin compounds, such as tributyltin (TBT) are typical environmental contaminants and suspected endocrine-disrupting chemical. The present study evaluates the estrogenic potential of this compound in vitro in ER (+) breast adenocarcinoma, MCF-7 cell line. Our data showed that tributyltin chloride (TBTCl) had agonistic activities for estrogen receptor-α (ER-α). Its estrogenic potential was checked using cell proliferation assay, aromatase assay, transactivation assay, and protein expression analysis. Low dose treatment of TBTCl had a proliferative effect on MCF-7 cells and resulted in up-regulation of aromatase enzyme activity and enhanced estradiol production in MCF-7 cells. Immunofluorescence staining showed translocation of ER-α from cytoplasm to nucleus and increased expression of ER-α, 3β-HSD and aromatase on treatment with increasing doses of TBTCl. Further, to decipher the probable signaling pathways involved in its action, the MCF-7 cells were transfected with different pathway dependent luciferase reporter plasmids (CRE, SRE, NF-κB and AP1). A significant increase in CRE and SRE and decrease in NF-κB regulated pathway were observed (p<0.05). Our results thus showed that the activation of SRE by TBTCl may be due to ligand dependent ER-α activation of the MAPK pathway and increased phosphorylation of ERK. In summary, the present data suggests that low dose of tributyltin genomically and non-genomically augmented estrogen dependent signaling by targeting various pathways. Copyright © 2013 Elsevier Inc. All rights reserved.

Position- and polarity-dependent Hippo signaling regulates cell fates in preimplantation mouse embryos.

PubMed

Sasaki, Hiroshi

2015-12-01

During the preimplantation stage, mouse embryos establish two cell lineages by the time of early blastocyst formation: the trophectoderm (TE) and the inner cell mass (ICM). Historical models have proposed that the establishment of these two lineages depends on the cell position within the embryo (e.g., the positional model) or cell polarization along the apicobasal axis (e.g., the polarity model). Recent findings have revealed that the Hippo signaling pathway plays a central role in the cell fate-specification process: active and inactive Hippo signaling in the inner and outer cells promote ICM and TE fates, respectively. Intercellular adhesion activates, while apicobasal polarization suppresses Hippo signaling, and a combination of these processes determines the spatially regulated activation of the Hippo pathway in 32-cell-stage embryos. Therefore, there is experimental evidence in favor of both positional and polarity models. At the molecular level, phosphorylation of the Hippo-pathway component angiomotin at adherens junctions (AJs) in the inner (apolar) cells activates the Lats protein kinase and triggers Hippo signaling. In the outer cells, however, cell polarization sequesters Amot from basolateral AJs and suppresses activation of the Hippo pathway. Other mechanisms, including asymmetric cell division and Notch signaling, also play important roles in the regulation of embryonic development. In this review, I discuss how these mechanisms cooperate with the Hippo signaling pathway during cell fate-specification processes. Copyright © 2015 Elsevier Ltd. All rights reserved.

Activation of cAMP-dependent signaling pathway induces mouse organic anion transporting polypeptide 2 expression.

PubMed

Chen, Chuan; Cheng, Xingguo; Dieter, Matthew Z; Tanaka, Yuji; Klaassen, Curtis D

2007-04-01

Rodent Oatp2 is a hepatic uptake transporter for such compounds as cardiac glycosides. In the present study, we found that fasting resulted in a 2-fold induction of Oatp2 expression in liver of mice. Because the cAMP-protein kinase A (PKA) signaling pathway is activated during fasting, the role of this pathway in Oatp2 induction during fasting was examined. In Hepa-1c1c7 cells, adenylyl cyclase activator forskolin as well as two cellular membrane-permeable cAMP analogs, dibutyryl cAMP and 8-bromo-cAMP, induced Oatp2 mRNA expression in a time- and dose-dependent manner. These three chemicals induced reporter gene activity in cells transfected with a luciferase reporter gene construct containing a 7.6-kilobase (kb) 5'-flanking region of mouse Oatp2. Transient transfection of cells with 5'-deletion constructs derived from the 7.6-kb Oatp2 promoter reporter gene construct, as well as 7.6-kb constructs in which a consensus cAMP response element (CRE) half-site CGTCA (-1808/-1804 bp) was mutated or deleted, confirms that this CRE site was required for the induction of luciferase activity by forskolin. Luciferase activity driven by the Oatp2 promoter containing this CRE site was induced in cells cotransfected with a plasmid encoding the protein kinase A catalytic subunit. Cotransfection of cells with a plasmid encoding the dominant-negative CRE binding protein (CREB) completely abolished the inducibility of the reporter gene activity by forskolin. In conclusion, induction of Oatp2 expression in liver of fasted mice may be caused by activation of the cAMP-dependent signaling pathway, with the CRE site (-1808/-1804) and CREB being the cis- and trans-acting factors mediating the induction, respectively.

RAE1 ligands for the NKG2D receptor are regulated by STING-dependent DNA sensor pathways in lymphoma.

PubMed

Lam, Adeline R; Bert, Nina Le; Ho, Samantha Sw; Shen, Yu J; Tang, Li Fm; Xiong, Gordon M; Croxford, John L; Koo, Christine X; Ishii, Ken J; Akira, Shizuo; Raulet, David H; Gasser, Stephan

2014-04-15

The immunoreceptor NKG2D originally identified in natural killer (NK) cells recognizes ligands that are upregulated on tumor cells. Expression of NKG2D ligands (NKG2DL) is induced by the DNA damage response (DDR), which is often activated constitutively in cancer cells, revealing them to NK cells as a mechanism of immunosurveillance. Here, we report that the induction of retinoic acid early transcript 1 (RAE1) ligands for NKG2D by the DDR relies on a STING-dependent DNA sensor pathway involving the effector molecules TBK1 and IRF3. Cytosolic DNA was detected in lymphoma cell lines that express RAE1 and its occurrence required activation of the DDR. Transfection of DNA into ligand-negative cells was sufficient to induce RAE1 expression. Irf3(+/-);Eμ-Myc mice expressed lower levels of RAE1 on tumor cells and showed a reduced survival rate compared with Irf3(+/+);Eμ-Myc mice. Taken together, our results suggest that genomic damage in tumor cells leads to activation of STING-dependent DNA sensor pathways, thereby activating RAE1 and enabling tumor immunosurveillance. ©2014 AACR.

Stress Inducibility of SIRT1 and Its Role in Cytoprotection and Cancer

PubMed Central

Raynes, Rachel; Brunquell, Jessica

2013-01-01

Cells must continuously respond to stressful insults via the upregulation of cytoprotective pathways. The longevity factor and deacetylase SIRT1 plays a critical role in coordinating this cellular response to stress. SIRT1 activity and levels are regulated by cellular stressors, including metabolic, genotoxic, oxidative, and proteotoxic stress. As a stress sensor, SIRT1 impacts cell survival by deacetylating substrate proteins to drive the cell towards a cytoprotective pathway. Extreme stress conditions, however, can cause SIRT1 to lead cells down an apoptotic pathway instead. SIRT1 is frequently dysregulated in cancer cells and has been characterized to have a dual role as both an oncogene and a tumor suppressor, likely due to its pivotal function in regulating cytoprotection. Recently, the ability of SIRT1 to regulate HSF1-dependent induction of the heat shock response has highlighted another pathway through which SIRT1 can modulate cytoprotection. Activation of HSF1 results in the production of cytoprotective chaperones that can facilitate the transformed phenotype of cancer cells. In this review, we discuss the stress-dependent regulation of SIRT1. We highlight the role of SIRT1 in stress management and cytoprotection and emphasize SIRT1-dependent activation of HSF1 as a potential mechanism for cancer promotion. PMID:24020008

A non-canonical RNA degradation pathway suppresses RNAi-dependent epimutations in the human fungal pathogen Mucor circinelloides.

PubMed

Calo, Silvia; Nicolás, Francisco E; Lee, Soo Chan; Vila, Ana; Cervantes, Maria; Torres-Martinez, Santiago; Ruiz-Vazquez, Rosa M; Cardenas, Maria E; Heitman, Joseph

2017-03-01

Mucorales are a group of basal fungi that includes the casual agents of the human emerging disease mucormycosis. Recent studies revealed that these pathogens activate an RNAi-based pathway to rapidly generate drug-resistant epimutant strains when exposed to stressful compounds such as the antifungal drug FK506. To elucidate the molecular mechanism of this epimutation pathway, we performed a genetic analysis in Mucor circinelloides that revealed an inhibitory role for the non-canonical RdRP-dependent Dicer-independent silencing pathway, which is an RNAi-based mechanism involved in mRNA degradation that was recently identified. Thus, mutations that specifically block the mRNA degradation pathway, such as those in the genes r3b2 and rdrp3, enhance the production of drug resistant epimutants, similar to the phenotype previously described for mutation of the gene rdrp1. Our genetic analysis also revealed two new specific components of the epimutation pathway related to the quelling induced protein (qip) and a Sad-3-like helicase (rnhA), as mutations in these genes prevented formation of drug-resistant epimutants. Remarkably, drug-resistant epimutant production was notably increased in M. circinelloides f. circinelloides isolates from humans or other animal hosts. The host-pathogen interaction could be a stressful environment in which the phenotypic plasticity provided by the epimutant pathway might provide an advantage for these strains. These results evoke a model whereby balanced regulation of two different RNAi pathways is determined by the activation of the RNAi-dependent epimutant pathway under stress conditions, or its repression when the regular maintenance of the mRNA degradation pathway operates under non-stress conditions.

c-Met must translocate to the nucleus to initiate calcium signals.

PubMed

Gomes, Dawidson A; Rodrigues, Michele A; Leite, M Fatima; Gomez, Marcus V; Varnai, Peter; Balla, Tamas; Bennett, Anton M; Nathanson, Michael H

2008-02-15

Hepatocyte growth factor (HGF) is important for cell proliferation, differentiation, and related activities. HGF acts through its receptor c-Met, which activates downstream signaling pathways. HGF binds to c-Met at the plasma membrane, where it is generally believed that c-Met signaling is initiated. Here we report that c-Met rapidly translocates to the nucleus upon stimulation with HGF. Ca(2+) signals that are induced by HGF result from phosphatidylinositol 4,5-bisphosphate hydrolysis and inositol 1,4,5-trisphosphate formation within the nucleus rather than within the cytoplasm. Translocation of c-Met to the nucleus depends upon the adaptor protein Gab1 and importin beta1, and formation of Ca(2+) signals in turn depends upon this translocation. HGF may exert its particular effects on cells because it bypasses signaling pathways in the cytoplasm to directly activate signaling pathways in the nucleus.

17betaE2 promotes cell proliferation in endometriosis by decreasing PTEN via NFkappaB-dependent pathway.

PubMed

Zhang, Hui; Zhao, Xingbo; Liu, Shu; Li, Jijun; Wen, Zeqing; Li, Mingjiang

2010-04-12

The objective of this study was to explore the mechanism of phosphatase and tensin homolog (PTEN) loss in endometriosis. We found that aberrant PTEN expression and mitogen-activated protein kinases (MAPK)/ERK, phosphoinositide 3-kinase (PI3K)/AKt, and nuclear factor-kappaB (NFkappaB) signaling overactivities coexisted in endometriosis. In vitro, 17beta-estradiol rapidly activated the 3 pathways in endometriotic cells and specific inhibitions on the 3 pathways respectively blocked 17beta-estradiol-induced cell proliferation. 17beta-estradiol suppressed PTEN transcription and expression in endometriotic cells which was abolished by specific NFkappaB inhibition. Total/nuclear PTEN-loss and MAPK/ERK, PI3K/AKt, and NFkappaB signal overactivities coexist in endometriosis. In vitro, 17beta-estradiol can promotes cell proliferation in endometriosis by activating PI3K/AKt pathway via an NFkappaB/PTEN-dependent pathway. For the first time we propose the possibility of the presence of a positive feedback-loop: 17beta-estradiol-->high NFkappaB-->low PTEN-->high PI3K-->high NFkappaB, in endometriosis, which may finally promote the proliferation of ectopic endometrial epithelial cells and in turn contributes to the progression of the disease.

Depletion of ribosomal protein L37 occurs in response to DNA damage and activates p53 through the L11/MDM2 pathway

PubMed Central

Llanos, Susana; Serrano, Manuel

2013-01-01

Perturbation of ribosomal biogenesis has recently emerged as a relevant p53-activating pathway. This pathway can be initiated by depletion of certain ribosomal proteins, which is followed by the binding and inhibition of MDM2 by a different subset of ribosomal proteins that includes L11. Here, we report that depletion of L37 leads to cell cycle arrest in a L11- and p53-dependent manner. DNA damage can initiate ribosomal stress, although little is known about the mechanisms involved. We have found that some genotoxic insults, namely UV light and cisplatin, lead to proteasomal degradation of L37 in the nucleoplasm and to the ensuing L11-dependent stabilization of p53. Moreover, ectopic L37 overexpression can attenuate the DNA damage response mediated by p53. These results support the concept that DNA damage-induced proteasomal degradation of L37 constitutes a mechanistic link between DNA damage and the ribosomal stress pathway, and is a relevant contributing signaling pathway for the activation of p53 in response to DNA damage. PMID:20935493

Activation of c-Raf-1 kinase signal transduction pathway in alpha(7) integrin-deficient mice.

PubMed

Saher, G; Hildt, E

1999-09-24

Integrin alpha(7)-deficient mice develop a novel form of muscular dystrophy. Here we report that deficiency of alpha(7) integrin causes an activation of the c-Raf-1/mitogen-activated protein (MAP) 2 kinase signal transduction pathway in muscle cells. The observed activation of c-Raf-1/MAP2 kinases is a specific effect, because the alpha(7) integrin deficiency does not cause unspecific stress as determined by measurement of the Hsp72/73 level and activity of the JNK2 kinase. Because an increased level of activated FAK was found in muscle of alpha(7) integrin-deficient mice, the activation of c-Raf-1 kinase is triggered most likely by an integrin-dependent pathway. In accordance with this, in the integrin alpha(7)-deficient mice, part of the integrin beta(1D) variant in muscle is replaced by the beta(1A) variant, which permits the FAK activation. A recent report describes that integrin activity can be down-modulated by the c-Raf-1/MAP2 kinase pathway. Specific activation of the c-Raf-1/MAP2 kinases by cell-permeable peptides in skeletal muscle of rabbits causes degeneration of muscle fibers. Therefore, we conclude that in alpha(7) integrin-deficient mice, the continuous activation of c-Raf-1 kinase causes a permanent reduction of integrin activity diminishing integrin-dependent cell-matrix interactions and thereby contributing to the development of the dystrophic phenotype.

Lipopolysaccharide regulation of intestinal tight junction permeability is mediated by TLR-4 signal transduction pathway activation of FAK and MyD88

PubMed Central

Guo, Shuhong; Nighot, Meghali; Al-Sadi, Rana; Alhmoud, Tarik; Nighot, Prashant; Ma, Thomas Y.

2015-01-01

Gut-derived bacterial lipopolysaccharides (LPS) play an essential role in inducing intestinal and systemic inflammatory responses and have been implicated as a pathogenic factor of necrotizing enterocolitis (NEC) and inflammatory bowel disease (IBD). The defective intestinal tight junction (TJ) barrier has been shown to be an important factor contributing to the development of intestinal inflammation. LPS, at physiological concentrations, cause an increase in intestinal tight junction permeability (TJP) via a TLR-4 dependent process; however the intracellular mechanisms that mediate LPS regulation of intestinal TJP remain unclear. The aim of this study was to investigate the adaptor proteins and the signaling interactions that mediate LPS modulation of intestinal TJ barrier using an in-vitro and in-vivo model system. LPS caused a TLR-4 dependent activation of membrane-associated adaptor protein FAK in Caco-2 monolayers. LPS caused an activation of both MyD88-dependent and –independent pathways. SiRNA silencing of MyD88 prevented LPS-induced increase in TJP. LPS caused a MyD88-dependent activation of IRAK4. TLR-4, FAK and MyD88 were co-localized. SiRNA silencing of TLR-4 inhibited TLR-4 associated FAK activation; and FAK knockdown prevented MyD88 activation. In-vivo studies also confirmed that LPS-induced increase in mouse intestinal permeability was associated with FAK and MyD88 activation; knockdown of intestinal epithelial FAK prevented LPS-induced increase in intestinal permeability. Additionally, high dose LPS-induced intestinal inflammation was also dependent on TLR-4/FAK/MyD88 signal-transduction axis. Our data show for the first time that LPS-induced increase in intestinal TJP and intestinal inflammation was regulated by TLR-4 dependent activation of FAK-MyD88-IRAK4 signaling pathway. PMID:26466961

Ginsenoside Rb1 protects against 6-hydroxydopamine-induced oxidative stress by increasing heme oxygenase-1 expression through an estrogen receptor-related PI3K/Akt/Nrf2-dependent pathway in human dopaminergic cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hwang, Yong Pil; College of Pharmacy, Chosun University, Gwangju; Jeong, Hye Gwang, E-mail: hgjeong@cnu.ac.k

Phytoestrogens are polyphenolic non-steroidal plant compounds with estrogen-like biological activity. Ginseng, the root of Panax ginseng C.A. Meyer (Araliaceae), is a popular traditional herbal medicine. Ginsenoside Rb1 (Rb1), an active component commonly found in ginseng root, is a phytoestrogen that exerts estrogen-like activity. In this study, we demonstrate that the phytoestrogen Rb1 inhibits 6-hydroxydopamine (6-OHDA)-induced oxidative injury via an ER-dependent Gbeta1/PI3K/Akt and heme oxygenase-1 (HO-1) pathway. Pretreatment of SH-SY5Y cells with Rb1 significantly reduced 6-OHDA-induced caspase-3 activation and subsequent cell death. Rb1 also up-regulated HO-1 expression, which conferred cytoprotection against 6-OHDA-induced oxidative injury. Moreover, Rb1 induced both Nrf2 nuclear translocation,more » which is upstream of HO-1 expression and PI3K activation, a pathway that is involved in induced Nrf2 nuclear translocation, HO-1 expression and cytoprotection. Also, Rb1-mediated increases in PI3K activation and HO-1 induction were reversed by co-treatment with ICI 182,780 and pertussis toxin. Taken together, these results suggest that Rb1 augments the cellular antioxidant defenses through ER-dependent HO-1 induction via the Gbeta1/PI3K/Akt-Nrf2 signaling pathway, thereby protecting cells from oxidative stress. Thus our study indicates that Rb1 has a partial cytoprotective role in dopaminergic cell culture systems.« less

4-Hydroxynonenal activates Src through a non-canonical pathway that involves EGFR/PTP1B

PubMed Central

Zhang, Hongqiao; Forman, Henry Jay

2015-01-01

Src, a non-receptor protein tyrosine kinase involved in many biological processes, can be activated through both redox-dependent and independent mechanisms. 4-Hydroxy-2-nonenal (HNE) is a lipid peroxidation product that is increased in pathophysiological conditions associated with Src activation. This study examined how HNE activates human c-Src. In the canonical pathway Src activation is initiated by dephosphorylation of pTyr530 followed by conformational change that causes Src auto-phosphorylation at Tyr419 and its activation. HNE increased Src activation in both dose- and time-dependent manner, while it also increased Src phosphorylation at Tyr530 (pTyr530 Src), suggesting that HNE activated Src via a non-canonical mechanism. Protein tyrosine phosphatase 1B inhibitor (539741), at concentrations that increased basal pTyr530 Src, also increased basal Src activity and significantly reduced HNE-mediated Src activation. The EGFR inhibitor, AG1478, and EGFR silencing, abrogated HNE-mediated EGFR activation and inhibited basal and HNE-induced Src activity. In addition, AG1478 also eliminated the increase of basal Src activation by a PTP1B inhibitor. Taken together these data suggest that HNE can activate Src partly through a non-canonical pathway involving activation of EGFR and inhibition of PTP1B. PMID:26453921

Chronic methamphetamine exposure induces cardiac fas-dependent and mitochondria-dependent apoptosis.

PubMed

Liou, Cher-Ming; Tsai, Shiow-Chwen; Kuo, Chia-Hua; Williams, Timothy; Ting, Hua; Lee, Shin-Da

2014-06-01

Very limited information regarding the influence of chronic methamphetamine exposure on cardiac apoptosis is available. In this study, we evaluate whether chronic methamphetamine exposure will increase cardiac Fas-dependent (type I) and mitochondria-dependent (type II) apoptotic pathways. Thirty-two male Wistar rats at 3-4 months of age were randomly divided into a vehicle-treated group [phosphate-buffered saline (PBS) 0.5 ml SQ per day] and a methamphetamine-treated group (MA 10 mg/kg SQ per day) for 3 months. We report that after 3 months of exposure, abnormal myocardial architecture, more minor cardiac fibrosis and cardiac TUNEL-positive apoptotic cells were observed at greater frequency in the MA group than in the PBS group. Protein levels of TNF-α, Fas ligand, Fas receptor, Fas-associated death domain, activated caspase-8, and activated caspase-3 (Fas-dependent apoptosis) extracted from excised hearts were significantly increased in the MA group, compared to the PBS group. Protein levels of cardiac Bak, t-Bid, Bak to Bcl-xL ratio, activated caspase-9, and activated caspase-3 (mitochondria-dependent apoptosis) were significantly increased in the MA group, compared with the PBS group. The results from this study reveal that chronic methamphetamine exposure will activate cardiac Fas-dependent and mitochondria-dependent apoptotic pathways, which may indicate a possible mechanism for developing cardiac abnormalities in humans with chronic methamphetamine abuse.

Core pathways operating during methylotrophy of Bacillus methanolicus MGA3 and induction of a bacillithiol-dependent detoxification pathway upon formaldehyde stress.

PubMed

Müller, Jonas E N; Meyer, Fabian; Litsanov, Boris; Kiefer, Patrick; Vorholt, Julia A

2015-12-01

Bacillus methanolicus MGA3 is a model facultative methylotroph of interest for fundamental research and biotechnological applications. Previous research uncovered a number of pathways potentially involved in one-carbon substrate utilization. Here, we applied dynamic (13) C labeling to elucidate which of these pathways operate during growth on methanol and to uncover potentially new ones. B. methanolicus MGA3 uses the assimilatory and dissimilatory ribulose monophosphate (RuMP) cycles for conversion of the central but toxic intermediate formaldehyde. Additionally, the operation of two cofactor-dependent formaldehyde oxidation pathways with distinct roles was revealed. One is dependent on tri- and tetraglutamylated tetrahydrofolate (THF) and is involved in formaldehyde oxidation during growth on methanol. A second pathway was discovered that is dependent on bacillithiol, a thiol cofactor present also in other Bacilli where it is known to function in redox-homeostasis. We show that bacillithiol-dependent formaldehyde oxidation is activated upon an upshift in formaldehyde induced by a substrate switch from mannitol to methanol. The genes and the corresponding enzymes involved in the biosynthesis of bacillithiol were identified by heterologous production of bacillithiol in Escherichia coli. The presented results indicate metabolic plasticity of the methylotroph allowing acclimation to fluctuating intracellular formaldehyde concentrations. © 2015 John Wiley & Sons Ltd.

Nox4 NADPH oxidase-derived reactive oxygen species, via endogenous carbon monoxide, promote survival of brain endothelial cells during TNF-α-induced apoptosis

PubMed Central

Basuroy, Shyamali; Tcheranova, Dilyara; Bhattacharya, Sujoy; Leffler, Charles W.

2011-01-01

We investigated the role of reactive oxygen species (ROS) in promoting cell survival during oxidative stress induced by the inflammatory mediator tumor necrosis factor-α (TNF-α) in cerebral microvascular endothelial cells (CMVEC) from newborn piglets. Nox4 is the major isoform of NADPH oxidase responsible for TNF-α-induced oxidative stress and apoptosis in CMVEC. We present novel data that Nox4 NADPH oxidase-derived ROS also initiate a cell survival mechanism by increasing production of a gaseous antioxidant mediator carbon monoxide (CO) by constitutive heme oxygenase-2 (HO-2). TNF-α rapidly enhanced endogenous CO production in a superoxide- and NADPH oxidase-dependent manner in CMVEC with innate, but not with small interfering RNA (siRNA)-downregulated Nox4 activity. CORM-A1, a CO-releasing compound, inhibited Nox4-mediated ROS production and enhanced cell survival in TNF-α-challenged CMVEC. The ROS-induced CO-mediated survival mechanism requires functional interactions between the protein kinase B/Akt and extracellular signal-related kinase (ERK)/p38 MAPK signaling pathways activated by TNF-α. In Akt siRNA-transfected CMVEC and in cells with pharmacologically inhibited Akt, Erk1/2, and p38 mitogen-activated protein kinase (MAPK) activities, CORM-A1 was no longer capable of blocking Nox4 activation and apoptosis caused by TNF-α. Overall, Nox4 NADPH oxidase-derived ROS initiate both death and survival pathways in TNF-α-challenged CMVEC. The ROS-dependent cell survival pathway is mediated by an endogenous antioxidant CO, which inhibits Nox4 activation via a mechanism that includes Akt, ERK1/2, and p38 MAPK signaling pathways. The ability of CO to inhibit TNF-α-induced ERK1/2 and p38 MAPK activities in an Akt-dependent manner appears to be the key element in ROS-dependent survival of endothelial cells during TNF-α-mediated brain inflammatory disease. PMID:21123734

Activity-Dependent IGF-1 Exocytosis is Controlled by the Ca2+-Sensor Synaptotagmin-10

PubMed Central

Cao, Peng; Maximov, Anton; Südhof, Thomas C.

2011-01-01

Synaptotagmins Syt1, Syt2, Syt7, and Syt9 act as Ca2+-sensors for synaptic and neuroendocrine exocytosis, but the function of other synaptotagmins remains unknown. Here, we show that olfactory bulb neurons secrete IGF-1 by an activity-dependent pathway of exocytosis, and that Syt10 functions as the Ca2+-sensor that triggers IGF-1 exocytosis in these neurons. Deletion of Syt10 impaired activity-dependent IGF-1 secretion in olfactory bulb neurons, resulting in smaller neurons and an overall decrease in synapse numbers. Exogenous IGF-1 completely reversed the Syt10 knockout phenotype. Syt10 co-localized with IGF-1 in somatodendritic vesicles of olfactory bulb neurons, and Ca2+-binding to Syt10 caused these vesicles to undergo exocytosis, thereby secreting IGF-1. Thus, Syt10 controls a previously unrecognized pathway of Ca2+-dependent exocytosis that is spatially and temporally distinct from Ca2+-dependent synaptic vesicle exocytosis controlled by Syt1 in the same neurons, and two different synaptotagmins regulate distinct Ca2+-dependent membrane fusion reactions during exocytosis in the same neuron. PMID:21496647

Task-dependent activation of distinct fast and slow(er) motor pathways during motor imagery.

PubMed

Keller, Martin; Taube, Wolfgang; Lauber, Benedikt

2018-02-22

Motor imagery and actual movements share overlapping activation of brain areas but little is known about task-specific activation of distinct motor pathways during mental simulation of movements. For real contractions, it was demonstrated that the slow(er) motor pathways are activated differently in ballistic compared to tonic contractions but it is unknown if this also holds true for imagined contractions. The aim of the present study was to assess the activity of fast and slow(er) motor pathways during mentally simulated movements of ballistic and tonic contractions. H-reflexes were conditioned with transcranial magnetic stimulation at different interstimulus intervals to assess the excitability of fast and slow(er) motor pathways during a) the execution of tonic and ballistic contractions, b) motor imagery of these contraction types, and c) at rest. In contrast to the fast motor pathways, the slow(er) pathways displayed a task-specific activation: for imagined ballistic as well as real ballistic contractions, the activation was reduced compared to rest whereas enhanced activation was found for imagined tonic and real tonic contractions. This study provides evidence that the excitability of fast and slow(er) motor pathways during motor imagery resembles the activation pattern observed during real contractions. The findings indicate that motor imagery results in task- and pathway-specific subliminal activation of distinct subsets of neurons in the primary motor cortex. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Cyanidin-3-O-β-glucoside regulates fatty acid metabolism via an AMP-activated protein kinase-dependent signaling pathway in human HepG2 cells

PubMed Central

2012-01-01

Background Hepatic metabolic derangements are key components in the development of fatty liver disease. AMP-activated protein kinase (AMPK) plays a central role in controlling hepatic lipid metabolism through modulating the downstream acetyl CoA carboxylase (ACC) and carnitine palmitoyl transferase 1 (CPT-1) pathway. In this study, cyanidin-3-O-β-glucoside (Cy-3-g), a typical anthocyanin pigment was used to examine its effects on AMPK activation and fatty acid metabolism in human HepG2 hepatocytes. Results Anthocyanin Cy-3-g increased cellular AMPK activity in a calmodulin kinase kinase dependent manner. Furthermore, Cy-3-g substantially induced AMPK downstream target ACC phosphorylation and inactivation, and then decreased malonyl CoA contents, leading to stimulation of CPT-1 expression and significant increase of fatty acid oxidation in HepG2 cells. These effects of Cy-3-g are largely abolished by pharmacological and genetic inhibition of AMPK. Conclusion This study demonstrates that Cy-3-g regulates hepatic lipid homeostasis via an AMPK-dependent signaling pathway. Targeting AMPK activation by anthocyanin may represent a promising approach for the prevention and treatment of obesity-related nonalcoholic fatty liver disease. PMID:22243683

Sorafenib induces cathepsin B-mediated apoptosis of bladder cancer cells by regulating the Akt/PTEN pathway. The Akt inhibitor, perifosine, enhances the sorafenib-induced cytotoxicity against bladder cancer cells

PubMed Central

Amantini, Consuelo; Morelli, Maria Beatrice; Santoni, Matteo; Soriani, Alessandra; Cardinali, Claudio; Farfariello, Valerio; Eleuteri, Anna Maria; Bonfili, Laura; Mozzicafreddo, Matteo; Nabissi, Massimo; Cascinu, Stefano; Santoni, Giorgio

2015-01-01

Sorafenib, a tyrosine kinase inhibitor, has been demonstrated to exert anti-tumor effects. However, the molecular mechanisms underlying its effects on bladder cancer remain unknown. Here, we evaluated the mechanisms responsible for the sorafenib-induced anti-tumor effects on 5637 and T24 bladder cancer cells. We demonstrated that sorafenib reduces cell viability, stimulates lysosome permeabilization and induces apoptosis of bladder cancer cells. These effects are dependent by the activation of cathepsin B released from lysosomes. The sorafenib-increased cathepsin B activity induced the proteolysis of Bid into tBid that stimulates the intrinsic pathway of apoptosis characterized by mitochondrial membrane depolarization, oxygen radical generation and cytochrome c release. Moreover, we found that cathepsin B enzymatic activity, induced by sorafenib, is dependent on its dephosphorylation via PTEN activation and Akt inactivation. Pretreatment with orthovanadate rescued bladder cancer cells from apoptosis. In addition, the Akt inhibitor perifosine increased the sensitivity of bladder cancer cells to sorafenib-induced cytotoxicity. Overall, our results show that apoptotic cell death induced by sorafenib in bladder cancer cells is dependent on cathepsin B activity and involved PTEN and Akt signaling pathways. The Akt inhibitor perifosine increased the cytotoxic effects of sorafenib in bladder cancer cells. PMID:26097873

Hydrogen peroxide inhibits Ca2+-dependent chloride secretion across colonic epithelial cells via distinct kinase signaling pathways and ion transport proteins

PubMed Central

Chappell, Alfred E.; Bunz, Michael; Smoll, Eric; Dong, Hui; Lytle, Christian; Barrett, Kim E.; McCole, Declan F.

2018-01-01

Reactive oxygen species (ROS) are key mediators in a number of inflammatory conditions, including inflammatory bowel disease (IBD). ROS, including hydrogen peroxide (H2O2), modulate intestinal epithelial ion transport and are believed to contribute to IBD-associated diarrhea. Intestinal crypt fluid secretion, driven by electrogenic Cl− secretion, hydrates and sterilizes the crypt, thus reducing bacterial adherence. Here, we show that pathophysiological concentrations of H2O2 inhibit Ca2+-dependent Cl− secretion across T84 colonic epithelial cells by elevating cytosolic Ca2+, which contributes to activation of two distinct signaling pathways. One involves recruitment of the Ca2+-responsive kinases, Src and Pyk-2, as well as extracellular signal-regulated kinase (ERK). A separate pathway recruits p38 MAP kinase and phosphoinositide 3-kinase (PI3-K) signaling. The ion transport response to Ca2+-dependent stimuli is mediated in part by K+ efflux through basolateral K+ channels and Cl− uptake by the Na+-K+-2Cl− cotransporter, NKCC1. We demonstrate that H2O2 inhibits Ca2+-dependent basolateral K+ efflux and also inhibits NKCC1 activity independently of inhibitory effects on apical Cl− conductance. Thus, we have demonstrated that H2O2 inhibits Ca2+-dependent Cl− secretion through multiple negative regulatory signaling pathways and inhibition of specific ion transporters. These findings increase our understanding of mechanisms by which inflammation disturbs intestinal epithelial function and contributes to intestinal pathophysiology.—Chappell, A. E., Bunz, M., Smoll, E., Dong, H., Lytle, C., Barrett, K. E., McCole, D. F. Hydrogen peroxide inhibits Ca2+-dependent chloride secretion across colonic epithelial cells via distinct kinase signaling pathways and ion transport proteins. FASEB J. 22, 000–000 (2008) PMID:18211955

Ca2+/calmodulin-dependent transcriptional pathways: potential mediators of skeletal muscle growth and development.

PubMed

Al-Shanti, Nasser; Stewart, Claire E

2009-11-01

The loss of muscle mass with age and disuse has a significant impact on the physiological and social well-being of the aged; this is an increasingly important problem as the population becomes skewed towards older age. Exercise has psychological benefits but it also impacts on muscle protein synthesis and degradation, increasing muscle tissue volume in both young and older individuals. Skeletal muscle hypertrophy involves an increase in muscle mass and cross-sectional area and associated increased myofibrillar protein content. Attempts to understand the molecular mechanisms that underlie muscle growth, development and maintenance, have focused on characterising the molecular pathways that initiate, maintain and regenerate skeletal muscle. Such understanding may aid in improving targeted interventional therapies for age-related muscle loss and muscle wasting associated with diseases. Two major routes through which skeletal muscle development and growth are regulated are insulin-like growth factor I (IGF-I) and Ca(2+)/calmodulin-dependent transcriptional pathways. Many reviews have focused on understanding the signalling pathways of IGF-I and its receptor, which govern skeletal muscle hypertrophy. However, alternative molecular signalling pathways such as the Ca(2+)/calmodulin-dependent transcriptional pathways should also be considered as potential mediators of muscle growth. These latter pathways have received relatively little attention and the purpose herein is to highlight the progress being made in the understanding of these pathways and associated molecules: calmodulin, calmodulin kinases (CaMKs), calcineurin and nuclear factor of activated T-cell (NFAT), which are involved in skeletal muscle regulation. We describe: (1) how conformational changes in the Ca(2+) sensor calmodulin result in the exposure of binding pockets for the target proteins (CaMKs and calcineurin). (2) How Calmodulin consequently activates either the Ca(2+)/calmodulin-dependent kinases pathways (via CaMKs) or calmodulin-dependent serine/threonine phosphatases (via calcineurin). (3) How calmodulin kinases alter transcription in the nucleus through the phosphorylation, deactivation and translocation of histone deacetylase 4 (HDAC4) from the nucleus to the cytoplasm. (4) How calcineurin transmits signals to the nucleus through the dephosphorylation and translocation of NFAT from the cytoplasm to the nucleus.

IL-1β-induced and p38MAPK-dependent activation of the mitogen-activated protein kinase-activated protein kinase 2 (MK2) in hepatocytes: Signal transduction with robust and concentration-independent signal amplification

PubMed Central

Kulawik, Andreas; Engesser, Raphael; Ehlting, Christian; Raue, Andreas; Albrecht, Ute; Hahn, Bettina; Lehmann, Wolf-Dieter; Gaestel, Matthias; Klingmüller, Ursula; Häussinger, Dieter; Timmer, Jens; Bode, Johannes G.

2017-01-01

The IL-1β induced activation of the p38MAPK/MAPK-activated protein kinase 2 (MK2) pathway in hepatocytes is important for control of the acute phase response and regulation of liver regeneration. Many aspects of the regulatory relevance of this pathway have been investigated in immune cells in the context of inflammation. However, very little is known about concentration-dependent activation kinetics and signal propagation in hepatocytes and the role of MK2. We established a mathematical model for IL-1β-induced activation of the p38MAPK/MK2 pathway in hepatocytes that was calibrated to quantitative data on time- and IL-1β concentration-dependent phosphorylation of p38MAPK and MK2 in primary mouse hepatocytes. This analysis showed that, in hepatocytes, signal transduction from IL-1β via p38MAPK to MK2 is characterized by strong signal amplification. Quantification of p38MAPK and MK2 revealed that, in hepatocytes, at maximum, 11.3% of p38MAPK molecules and 36.5% of MK2 molecules are activated in response to IL-1β. The mathematical model was experimentally validated by employing phosphatase inhibitors and the p38MAPK inhibitor SB203580. Model simulations predicted an IC50 of 1–1.2 μm for SB203580 in hepatocytes. In silico analyses and experimental validation demonstrated that the kinase activity of p38MAPK determines signal amplitude, whereas phosphatase activity affects both signal amplitude and duration. p38MAPK and MK2 concentrations and responsiveness toward IL-1β were quantitatively compared between hepatocytes and macrophages. In macrophages, the absolute p38MAPK and MK2 concentration was significantly higher. Finally, in line with experimental observations, the mathematical model predicted a significantly higher half-maximal effective concentration for IL-1β-induced pathway activation in macrophages compared with hepatocytes, underscoring the importance of cell type-specific differences in pathway regulation. PMID:28223354

Two distinct roles of mitogen-activated protein kinases in platelets and a novel Rac1-MAPK–dependent integrin outside-in retractile signaling pathway

PubMed Central

Flevaris, Panagiotis; Li, Zhenyu; Zhang, Guoying; Zheng, Yi; Liu, Junling

2009-01-01

Mitogen-activated protein kinases (MAPK), p38, and extracellular stimuli-responsive kinase (ERK), are acutely but transiently activated in platelets by platelet agonists, and the agonist-induced platelet MAPK activation is inhibited by ligand binding to the integrin αIIbβ3. Here we show that, although the activation of MAPK, as indicated by MAPK phosphorylation, is initially inhibited after ligand binding to integrin αIIbβ3, integrin outside-insignaling results in a late but sustained activation of MAPKs in platelets. Furthermore, we show that the early agonist-induced MAPK activation and the late integrin-mediated MAPK activation play distinct roles in different stages of platelet activation. Agonist-induced MAPK activation primarily plays an important role in stimulating secretion of platelet granules, while integrin-mediated MAPK activation is important in facilitating clot retraction. The stimulatory role of MAPK in clot retraction is mediated by stimulating myosin light chain (MLC) phosphorylation. Importantly, integrin-dependent MAPK activation, MAPK-dependent MLC phosphorylation, and clot retraction are inhibited by a Rac1 inhibitor and in Rac1 knockout platelets, indicating that integrin-induced activation of MAPK and MLC and subsequent clot retraction is Rac1-dependent. Thus, our results reveal 2 different activation mechanisms of MAPKs that are involved in distinct aspects of platelet function and a novel Rac1-MAPK–dependent cell retractile signaling pathway. PMID:18957688

Thrombin increases hyposmotic taurine efflux and accelerates ICI-swell and RVD in 3T3 fibroblasts by a src-dependent EGFR transactivation.

PubMed

Vázquez-Juárez, E; Ramos-Mandujano, G; Lezama, R A; Cruz-Rangel, S; Islas, L D; Pasantes-Morales, H

2008-02-01

The present study in Swiss3T3 fibroblasts examines the effect of thrombin on hyposmolarity-induced osmolyte fluxes and RVD, and the contribution of the src/EGFR pathway. Thrombin (5 U/ml) added to a 30% hyposmotic medium markedly increased hyposmotic 3H-taurine efflux (285%), accelerated the volume-sensitive Cl- current (ICI-swell) and increased RVD rate. These effects were reduced (50-65%) by preventing the thrombin-induced intracellular Ca2+ [Ca2+]i rise with EGTA-AM, or with the phospholipase C (PLC) blocker U73122. Ca2+calmodulin (CaM) and calmodulin kinase II (CaMKII) also participate in this Ca2+-dependent pathway. Thrombin plus hyposmolarity increased src and EGFR phosphorylation, whose blockade by PP2 and AG1478, decreased by 30-50%, respectively, the thrombin effects on hyposmotic taurine efflux, ICI-swell and RVD. Ca2+- and src/EGFR-mediated pathways operate independently as shown by (1) the persistence of src and EGFR activation when [Ca2+]i rise is prevented and (2) the additive effect on taurine efflux, ICI-swell or RVD by simultaneous inhibition of the two pathways, which essentially suppressed these events. PLC-Ca2+- and src/EGFR-signaling pathways operate in the hyposmotic condition and because thrombin per se failed to increase taurine efflux and ICI-swell under isosmotic condition it seems that it is merely amplifying these previously activated mechanisms. The study shows that thrombin potentiates hyposmolarity-induced osmolyte fluxes and RVD by increasing src/EGFR-dependent signaling, in addition to the Ca2+-dependent pathway.

ADP-ribosylation Factor 6 (ARF6) Bidirectionally Regulates Dendritic Spine Formation Depending on Neuronal Maturation and Activity*

PubMed Central

Kim, Yoonju; Lee, Sang-Eun; Park, Joohyun; Kim, Minhyung; Lee, Boyoon; Hwang, Daehee; Chang, Sunghoe

2015-01-01

Recent studies have reported conflicting results regarding the role of ARF6 in dendritic spine development, but no clear answer for the controversy has been suggested. We found that ADP-ribosylation factor 6 (ARF6) either positively or negatively regulates dendritic spine formation depending on neuronal maturation and activity. ARF6 activation increased the spine formation in developing neurons, whereas it decreased spine density in mature neurons. Genome-wide microarray analysis revealed that ARF6 activation in each stage leads to opposite patterns of expression of a subset of genes that are involved in neuronal morphology. ARF6-mediated Rac1 activation via the phospholipase D pathway is the coincident factor in both stages, but the antagonistic RhoA pathway becomes involved in the mature stage. Furthermore, blocking neuronal activity in developing neurons using tetrodotoxin or enhancing the activity in mature neurons using picrotoxin or chemical long term potentiation reversed the effect of ARF6 on each stage. Thus, activity-dependent dynamic changes in ARF6-mediated spine structures may play a role in structural plasticity of mature neurons. PMID:25605715

PIK3CA dependence and sensitivity to therapeutic targeting in urothelial carcinoma.

PubMed

Ross, R L; McPherson, H R; Kettlewell, L; Shnyder, S D; Hurst, C D; Alder, O; Knowles, M A

2016-07-28

Many urothelial carcinomas (UC) contain activating PIK3CA mutations. In telomerase-immortalized normal urothelial cells (TERT-NHUC), ectopic expression of mutant PIK3CA induces PI3K pathway activation, cell proliferation and cell migration. However, it is not clear whether advanced UC tumors are PIK3CA-dependent and whether PI3K pathway inhibition is a good therapeutic option in such cases. We used retrovirus-mediated delivery of shRNA to knock down mutant PIK3CA in UC cell lines and assessed effects on pathway activation, cell proliferation, migration and tumorigenicity. The effect of the class I PI3K inhibitor GDC-0941 was assessed in a panel of UC cell lines with a range of known molecular alterations in the PI3K pathway. Specific knockdown of PIK3CA inhibited proliferation, migration, anchorage-independent growth and in vivo tumor growth of cells with PIK3CA mutations. Sensitivity to GDC-0941 was dependent on hotspot PIK3CA mutation status. Cells with rare PIK3CA mutations and co-occurring TSC1 or PTEN mutations were less sensitive. Furthermore, downstream PI3K pathway alterations in TSC1 or PTEN or co-occurring AKT1 and RAS gene mutations were associated with GDC-0941 resistance. Mutant PIK3CA is a potent oncogenic driver in many UC cell lines and may represent a valuable therapeutic target in advanced bladder cancer.

13C nuclear magnetic resonance detection of interactions of serine hydroxymethyltransferase with C1-tetrahydrofolate synthase and glycine decarboxylase complex activities in Arabidopsis.

PubMed Central

Prabhu, V; Chatson, K B; Abrams, G D; King, J

1996-01-01

In C3 plants, serine synthesis is associated with photorespiratory glycine metabolism involving the tetrahydrofolate (THF)-dependent activities of the glycine decarboxylase complex (GDC) and serine hydroxymethyl transferase (SHMT). Alternatively, THF-dependent serine synthesis can occur via the C1-THF synthase/SHMT pathway. We used 13C nuclear magnetic resonance to examine serine biosynthesis by these two pathways in Arabidopsis thaliana (L.) Heynh. Columbia wild type. We confirmed the tight coupling of the GDC/ SHMT system and observed directly in a higher plant the flux of formate through the C1-THF synthase/SHMT system. The accumulation of 13C-enriched serine over 24 h from the GDC/SHMT activities was 4-fold greater than that from C1-THF synthase/SHMT activities. Our experiments strongly suggest that the two pathways operate independently in Arabidopsis. Plants exposed to methotrexate and sulfanilamide, powerful inhibitors of THF biosynthesis, reduced serine synthesis by both pathways. The results suggest that continuous supply of THF is essential to maintain high rates of serine metabolism. Nuclear magnetic resonance is a powerful tool for the examination of THF-mediated metabolism in its natural cellular environment. PMID:8819325

An Eph receptor sperm-sensing control mechanism for oocyte meiotic maturation in Caenorhabditis elegans.

PubMed

Miller, Michael A; Ruest, Paul J; Kosinski, Mary; Hanks, Steven K; Greenstein, David

2003-01-15

During sexual reproduction in most animals, oocytes arrest in meiotic prophase and resume meiosis (meiotic maturation) in response to sperm or somatic cell signals. Despite progress in delineating mitogen-activated protein kinase (MAPK) and CDK/cyclin activation pathways involved in meiotic maturation, it is less clear how these pathways are regulated at the cell surface. The Caenorhabditis elegans major sperm protein (MSP) signals oocytes, which are arrested in meiotic prophase, to resume meiosis and ovulate. We used DNA microarray data and an in situ binding assay to identify the VAB-1 Eph receptor protein-tyrosine kinase as an MSP receptor. We show that VAB-1 and a somatic gonadal sheath cell-dependent pathway, defined by the CEH-18 POU-class homeoprotein, negatively regulate meiotic maturation and MAPK activation. MSP antagonizes these inhibitory signaling circuits, in part by binding VAB-1 on oocytes and sheath cells. Our results define a sperm-sensing control mechanism that inhibits oocyte maturation, MAPK activation, and ovulation when sperm are unavailable for fertilization. MSP-domain proteins are found in diverse animal taxa, where they may regulate contact-dependent Eph receptor signaling pathways.

Cross-Talk Between Mitochondrial Fusion and the Hippo Pathway in Controlling Cell Proliferation During Drosophila Development.

PubMed

Deng, Qiannan; Guo, Ting; Zhou, Xiu; Xi, Yongmei; Yang, Xiaohang; Ge, Wanzhong

2016-08-01

Cell proliferation and tissue growth depend on the coordinated regulation of multiple signaling molecules and pathways during animal development. Previous studies have linked mitochondrial function and the Hippo signaling pathway in growth control. However, the underlying molecular mechanisms are not fully understood. Here we identify a Drosophila mitochondrial inner membrane protein ChChd3 as a novel regulator for tissue growth. Loss of ChChd3 leads to tissue undergrowth and cell proliferation defects. ChChd3 is required for mitochondrial fusion and removal of ChChd3 increases mitochondrial fragmentation. ChChd3 is another mitochondrial target of the Hippo pathway, although it is only partially required for Hippo pathway-mediated overgrowth. Interestingly, lack of ChChd3 leads to inactivation of Hippo activity under normal development, which is also dependent on the transcriptional coactivator Yorkie (Yki). Furthermore, loss of ChChd3 induces oxidative stress and activates the JNK pathway. In addition, depletion of other mitochondrial fusion components, Opa1 or Marf, inactivates the Hippo pathway as well. Taken together, we propose that there is a cross-talk between mitochondrial fusion and the Hippo pathway, which is essential in controlling cell proliferation and tissue homeostasis in Drosophila. Copyright © 2016 by the Genetics Society of America.

Age-dependent changes of the antioxidant system in rat livers are accompanied by altered MAPK activation and a decline in motor signaling

PubMed Central

Yang, Wei; Burkhardt, Britta; Fischer, Luise; Beirow, Maja; Bork, Nadja; Wönne, Eva C.; Wagner, Cornelia; Husen, Bettina; Zeilinger, Katrin; Liu, Liegang; Nussler, Andreas K.

2015-01-01

Aging is characterized by a progressive decrease of cellular functions, because cells gradually lose their capacity to respond to injury. Increased oxidative stress is considered to be one of the major contributors to age-related changes in all organs including the liver. Our study has focused on elucidating whether important antioxidative enzymes, the mTOR pathway, and MAPKs exhibit age-dependent changes in the liver of rats during aging. We found an age-dependent increase of GSH in the cytosol and mitochondria. The aged liver showed an increased SOD enzyme activity, while the CAT enzyme activity decreased. HO-1 and NOS-2 gene expression was lower in adult rats, but up-regulated in aged rats. Western blot analysis revealed that SOD1, SOD2, GPx, GR, γ-GCL, and GSS were age-dependent up-regulated, while CAT remained constant. We also demonstrated that the phosphorylation of Akt, JNK, p38, and TSC2Ser1254 decreased while ERK1/2 and TSC2Thr1462 increased age-dependently. Furthermore, our data show that the mTOR pathway seems to be activated in livers of aged rats, and hence stimulating cell proliferation/regeneration, as confirmed by an age-dependent increase of PCNA and p-eIF4ESer209 protein expression. Our data may help to explain the fact that liver cells only proliferate in cases of necessity, like injury and damage. In summary, we have demonstrated that, age-dependent changes of the antioxidant system and stress-related signaling pathways occur in the livers of rats, which may help to better understand organ aging. PMID:27004051

Constitutive and ghrelin-dependent GHSR1a activation impairs CaV2.1 and CaV2.2 currents in hypothalamic neurons

PubMed Central

López Soto, Eduardo Javier; Agosti, Francina; Cabral, Agustina; Mustafa, Emilio Roman; Damonte, Valentina Martínez; Gandini, Maria Alejandra; Rodríguez, Silvia; Castrogiovanni, Daniel; Felix, Ricardo; Perelló, Mario

2015-01-01

The growth hormone secretagogue receptor type 1a (GHSR1a) has the highest known constitutive activity of any G protein–coupled receptor (GPCR). GHSR1a mediates the action of the hormone ghrelin, and its activation increases transcriptional and electrical activity in hypothalamic neurons. Although GHSR1a is present at GABAergic presynaptic terminals, its effect on neurotransmitter release remains unclear. The activities of the voltage-gated calcium channels, CaV2.1 and CaV2.2, which mediate neurotransmitter release at presynaptic terminals, are modulated by many GPCRs. Here, we show that both constitutive and agonist-dependent GHSR1a activity elicit a strong impairment of CaV2.1 and CaV2.2 currents in rat and mouse hypothalamic neurons and in a heterologous expression system. Constitutive GHSR1a activity reduces CaV2 currents by a Gi/o-dependent mechanism that involves persistent reduction in channel density at the plasma membrane, whereas ghrelin-dependent GHSR1a inhibition is reversible and involves altered CaV2 gating via a Gq-dependent pathway. Thus, GHSR1a differentially inhibits CaV2 channels by Gi/o or Gq protein pathways depending on its mode of activation. Moreover, we present evidence suggesting that GHSR1a-mediated inhibition of CaV2 attenuates GABA release in hypothalamic neurons, a mechanism that could contribute to neuronal activation through the disinhibition of postsynaptic neurons. PMID:26283199

Tissue plasminogen activator mediates amyloid-induced neurotoxicity via Erk1/2 activation

PubMed Central

Medina, Manel G; Ledesma, Maria Dolores; Domínguez, Jorge E; Medina, Miguel; Zafra, Delia; Alameda, Francesc; Dotti, Carlos G; Navarro, Pilar

2005-01-01

Tissue plasminogen activator (tPA) is the main activator of plasminogen into plasmin in the brain where it may have beneficial roles but also neurotoxic effects that could be plasmin dependent or not. Little is known about the substrates and pathways that mediate plasmin-independent tPA neurotoxicity. Here we show in primary hippocampal neurons that tPA promotes a catalytic-independent activation of the extracellular regulated kinase (Erk)1/2 signal transduction pathway through the N-methyl-D-aspartate receptor, G-proteins and protein kinase C. This results in GSK3 activation in a process that requires de novo synthesis of proteins, and leads to tau aberrant phosphorylation, microtubule destabilization and apoptosis. Similar effects are produced by amyloid aggregates in a tPA-dependent manner, as demonstrated by pharmacological treatments and in wt and tPA−/− mice neurons. Consistently, in Alzheimer's disease (AD) patients' brains, high levels of tPA colocalize with amyloid-rich areas, activated Erk1/2 and phosphorylated tau. This is the first demonstration of an intracellular pathway by which tPA triggers kinase activation, tau phosphorylation and neurotoxicity, suggesting a key role for this molecule in AD pathology. PMID:15861134

Glabridin induces apoptosis and cell cycle arrest in oral cancer cells through the JNK1/2 signaling pathway.

PubMed

Chen, Chang-Tai; Chen, Yi-Tzu; Hsieh, Yi-Hsien; Weng, Chia-Jui; Yeh, Jung-Chun; Yang, Shun-Fa; Lin, Chiao-Wen; Yang, Jia-Sin

2018-06-01

Glabridin, a flavonoid extracted from licorice (Glycyrrhiza glabra), possesses various biological properties, including anticancer activities. However, the effect of glabridin on oral cancer cell apoptosis and the underlying molecular mechanisms has not been elucidated. In this study, we demonstrated that glabridin treatment significantly inhibits cell proliferation in human oral cancer SCC-9 and SAS cell lines. Flow cytometric assays demonstrated that glabridin induced several features of apoptosis, such as sub-G1 phase cell increase and phosphatidylserine externalization. Furthermore, glabridin induced apoptosis dose-dependently in SCC-9 cells through caspase-3, -8, and -9 activation and poly (ADP-ribose) polymerase cleavage. Moreover, glabridin increased the phosphorylation of the extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase (JNK) pathways in a dose-dependent manner. Moreover, the inhibition of the JNK1/2 inhibitor significantly reversed the glabridin-induced activation of the caspase pathway. In conclusion, our findings suggest that glabridin induces oral cancer cell apoptosis through the JNK1/2 pathway and is a potential therapeutic agent for oral cancer. © 2018 Wiley Periodicals, Inc.

The bone morphogenetic protein axis is a positive regulator of skeletal muscle mass

PubMed Central

Chen, Justin L.; Qian, Hongwei; Liu, Yingying; Bernardo, Bianca C.; Beyer, Claudia; Watt, Kevin I.; Thomson, Rachel E.; Connor, Timothy; Turner, Bradley J.; McMullen, Julie R.; Larsson, Lars; McGee, Sean L.; Harrison, Craig A.

2013-01-01

Although the canonical transforming growth factor β signaling pathway represses skeletal muscle growth and promotes muscle wasting, a role in muscle for the parallel bone morphogenetic protein (BMP) signaling pathway has not been defined. We report, for the first time, that the BMP pathway is a positive regulator of muscle mass. Increasing the expression of BMP7 or the activity of BMP receptors in muscles induced hypertrophy that was dependent on Smad1/5-mediated activation of mTOR signaling. In agreement, we observed that BMP signaling is augmented in models of muscle growth. Importantly, stimulation of BMP signaling is essential for conservation of muscle mass after disruption of the neuromuscular junction. Inhibiting the phosphorylation of Smad1/5 exacerbated denervation-induced muscle atrophy via an HDAC4-myogenin–dependent process, whereas increased BMP–Smad1/5 activity protected muscles from denervation-induced wasting. Our studies highlight a novel role for the BMP signaling pathway in promoting muscle growth and inhibiting muscle wasting, which may have significant implications for the development of therapeutics for neuromuscular disorders. PMID:24145169

Effect of TPA and HTLV-1 Tax on BRCA1 and ERE controlled genes expression.

PubMed

Jabareen, Azhar; Abu-Jaafar, Aya; Abou-Kandil, Ammar; Huleihel, Mahmoud

2017-07-18

Interference with the expression and/or functions of the multifunctional tumor suppressor BRCA1 leads to a high risk of breast and ovarian cancers. BRCA1 expression is usually activated by the estrogen (E2) liganded ERα receptor. Activated ERα is considered as a potent transcription factor which activates various genes expression by 2 pathways. A classical pathway, ERα binds directly to E2-responsive elements (EREs) in the promoters of the responsive genes and a non-classical pathway where ERα indirectly binds with the appropriate gene promoter. In our previous study, HTLV-1Tax was found to strongly inhibit ERα induced BRCA1 expression while stimulating ERα induced ERE dependent genes. TPA is a strong PKC activator which found to induce the expression of HTLV-1. Here we examined the effect of TPA on the expression of BRCA1 and genes controlled by ERE region in MCF-7 cells and on Tax activity on these genes. Our results showed strong stimulatory effect of TPA on both BRCA1 and ERE expression without treatment with E2. Tax did not show any significant effect on these TPA activities. It seems that TPA activation of BRCA1 and ERE expression is dependent on PKC activity but not through the NFκB pathway. However, 53BP1 may be involved in this TPA activity because its overexpression significantly reduced the TPA stimulatory effect on BRCA1 and ERE expression. Additionally, our Chip assay results probably exclude possible involvement of ERα pathway in this TPA activity because TPA did not interfere with the binding of ERα to both BRCA1 promoter and ERE region.

A Protein Scaffold Coordinates SRC-Mediated JNK Activation in Response to Metabolic Stress.

PubMed

Kant, Shashi; Standen, Claire L; Morel, Caroline; Jung, Dae Young; Kim, Jason K; Swat, Wojciech; Flavell, Richard A; Davis, Roger J

2017-09-19

Obesity is a major risk factor for the development of metabolic syndrome and type 2 diabetes. How obesity contributes to metabolic syndrome is unclear. Free fatty acid (FFA) activation of a non-receptor tyrosine kinase (SRC)-dependent cJun NH 2 -terminal kinase (JNK) signaling pathway is implicated in this process. However, the mechanism that mediates SRC-dependent JNK activation is unclear. Here, we identify a role for the scaffold protein JIP1 in SRC-dependent JNK activation. SRC phosphorylation of JIP1 creates phosphotyrosine interaction motifs that bind the SH2 domains of SRC and the guanine nucleotide exchange factor VAV. These interactions are required for SRC-induced activation of VAV and the subsequent engagement of a JIP1-tethered JNK signaling module. The JIP1 scaffold protein, therefore, plays a dual role in FFA signaling by coordinating upstream SRC functions together with downstream effector signaling by the JNK pathway. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Interconnections between mRNA degradation and RDR-dependent siRNA production in mRNA turnover in plants.

PubMed

Tsuzuki, Masayuki; Motomura, Kazuki; Kumakura, Naoyoshi; Takeda, Atsushi

2017-03-01

Accumulation of an mRNA species is determined by the balance between the synthesis and the degradation of the mRNA. Individual mRNA molecules are selectively and actively degraded through RNA degradation pathways, which include 5'-3' mRNA degradation pathway, 3'-5' mRNA degradation pathway, and RNA-dependent RNA polymerase-mediated mRNA degradation pathway. Recent studies have revealed that these RNA degradation pathways compete with each other in mRNA turnover in plants and that plants have a hidden layer of non-coding small-interfering RNA production from a set of mRNAs. In this review, we summarize the current information about plant mRNA degradation pathways in mRNA turnover and discuss the potential roles of a novel class of the endogenous siRNAs derived from plant mRNAs.

Proteomic analysis of the signaling pathway mediated by the heterotrimeric Gα protein Pga1 of Penicillium chrysogenum.

PubMed

Carrasco-Navarro, Ulises; Vera-Estrella, Rosario; Barkla, Bronwyn J; Zúñiga-León, Eduardo; Reyes-Vivas, Horacio; Fernández, Francisco J; Fierro, Francisco

2016-10-06

The heterotrimeric Gα protein Pga1-mediated signaling pathway regulates the entire developmental program in Penicillium chrysogenum, from spore germination to the formation of conidia. In addition it participates in the regulation of penicillin biosynthesis. We aimed to advance the understanding of this key signaling pathway using a proteomics approach, a powerful tool to identify effectors participating in signal transduction pathways. Penicillium chrysogenum mutants with different levels of activity of the Pga1-mediated signaling pathway were used to perform comparative proteomic analyses by 2D-DIGE and LC-MS/MS. Thirty proteins were identified which showed differences in abundance dependent on Pga1 activity level. By modifying the intracellular levels of cAMP we could establish cAMP-dependent and cAMP-independent pathways in Pga1-mediated signaling. Pga1 was shown to regulate abundance of enzymes in primary metabolic pathways involved in ATP, NADPH and cysteine biosynthesis, compounds that are needed for high levels of penicillin production. An in vivo phosphorylated protein containing a pleckstrin homology domain was identified; this protein is a candidate for signal transduction activity. Proteins with possible roles in purine metabolism, protein folding, stress response and morphogenesis were also identified whose abundance was regulated by Pga1 signaling. Thirty proteins whose abundance was regulated by the Pga1-mediated signaling pathway were identified. These proteins are involved in primary metabolism, stress response, development and signal transduction. A model describing the pathways through which Pga1 signaling regulates different cellular processes is proposed.

Attenuation of Aβ{sub 25–35}-induced parallel autophagic and apoptotic cell death by gypenoside XVII through the estrogen receptor-dependent activation of Nrf2/ARE pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meng, Xiangbao; Wang, Min; Sun, Guibo, E-mail: sunguibo@126.com

Amyloid-beta (Aβ) has a pivotal function in the pathogenesis of Alzheimer's disease. To investigate Aβ neurotoxicity, we used an in vitro model that involves Aβ{sub 25–35}-induced cell death in the nerve growth factor-induced differentiation of PC12 cells. Aβ{sub 25–35} (20 μM) treatment for 24 h caused apoptotic cell death, as evidenced by significant cell viability reduction, LDH release, phosphatidylserine externalization, mitochondrial membrane potential disruption, cytochrome c release, caspase-3 activation, PARP cleavage, and DNA fragmentation in PC12 cells. Aβ{sub 25–35} treatment led to autophagic cell death, as evidenced by augmented GFP-LC3 puncta, conversion of LC3-I to LC3-II, and increased LC3-II/LC3-I ratio.more » Aβ{sub 25–35} treatment induced oxidative stress, as evidenced by intracellular ROS accumulation and increased production of mitochondrial superoxide, malondialdehyde, protein carbonyl, and 8-OHdG. Phytoestrogens have been proved to be protective against Aβ-induced neurotoxicity and regarded as relatively safe targets for AD drug development. Gypenoside XVII (GP-17) is a novel phytoestrogen isolated from Gynostemma pentaphyllum or Panax notoginseng. Pretreatment with GP-17 (10 μM) for 12 h increased estrogen response element reporter activity, activated PI3K/Akt pathways, inhibited GSK-3β, induced Nrf2 nuclear translocation, augmented antioxidant responsive element enhancer activity, upregulated heme oxygenase 1 (HO-1) expression and activity, and provided protective effects against Aβ{sub 25–35}-induced neurotoxicity, including oxidative stress, apoptosis, and autophagic cell death. In conclusion, GP-17 conferred protection against Aβ{sub 25–35}-induced neurotoxicity through estrogen receptor-dependent activation of PI3K/Akt pathways, inactivation of GSK-3β and activation of Nrf2/ARE/HO-1 pathways. This finding might provide novel insights into understanding the mechanism for neuroprotective effects of phytoestrogens or gypenosides. - Highlights: • GP-17 showed protection against Aβ{sub 25–35}-induced neurotoxicity. • The neuroprotective effects of GP-17 are dependent on estrogen receptors. • GP-17 activates Nrf2/ARE/HO-1 pathways. • GP-17 activates PI3K/Akt/GSK-3β pathways.« less

Estradiol targets T cell signaling pathways in human systemic lupus.

PubMed

Walters, Emily; Rider, Virginia; Abdou, Nabih I; Greenwell, Cindy; Svojanovsky, Stan; Smith, Peter; Kimler, Bruce F

2009-12-01

The major risk factor for developing systemic lupus erythematosus (SLE) is being female. The present study utilized gene profiles of activated T cells from females with SLE and healthy controls to identify signaling pathways uniquely regulated by estradiol that could contribute to SLE pathogenesis. Selected downstream pathway genes (+/- estradiol) were measured by real time polymerase chain amplification. Estradiol uniquely upregulated six pathways in SLE T cells that control T cell function including interferon-alpha signaling. Measurement of interferon-alpha pathway target gene expression revealed significant differences (p= 0.043) in DRIP150 (+/- estradiol) in SLE T cell samples while IFIT1 expression was bimodal and correlated moderately (r= 0.55) with disease activity. The results indicate that estradiol alters signaling pathways in activated SLE T cells that control T cell function. Differential expression of transcriptional coactivators could influence estrogen-dependent gene regulation in T cell signaling and contribute to SLE onset and disease pathogenesis.

Microbiota activates IMD pathway and limits Sindbis infection in Aedes aegypti.

PubMed

Barletta, Ana Beatriz Ferreira; Nascimento-Silva, Maria Clara L; Talyuli, Octávio A C; Oliveira, José Henrique M; Pereira, Luiza Oliveira Ramos; Oliveira, Pedro L; Sorgine, Marcos Henrique F

2017-02-23

Aedes aegypti is the main vector of important arboviruses such as dengue, Zika and chikungunya. During infections mosquitoes can activate the immune pathways Toll, IMD and JAK/STAT to limit pathogen replication. Here, we evaluate the immune response profile of Ae. aegypti against Sindbis virus (SINV). We analyzed gene expression of components of Toll, IMD and JAK/STAT pathways and showed that a blood meal and virus infection upregulated aaREL2 in a microbiota-dependent fashion, since this induction was prevented by antibiotic. The presence of the microbiota activates IMD and impaired the replication of SINV in the midgut. Constitutive activation of the IMD pathway, by Caspar depletion, leads to a decrease in microbiota levels and an increase in SINV loads. Together, these results suggest that a blood meal is able to activate innate immune pathways, through a nutrient induced growth of microbiota, leading to upregulation of aaREL2 and IMD activation. Microbiota levels seemed to have a reciprocal interaction, where the proliferation of the microbiota activates IMD pathway that in turn controls bacterial levels, allowing SINV replication in Ae. aegypti mosquitoes. The activation of the IMD pathway seems to have an indirect effect in SINV levels that is induced by the microbiota.

Involvement of the cGMP pathway in the osthole-facilitated glutamate release in rat hippocampal nerve endings.

PubMed

Lin, Tzu Yu; Lu, Cheng Wei; Huang, Wei-Jan; Wang, Su-Jane

2012-03-01

Osthole, an active constituent isolated from Cnidium monnieri (L.) Cusson, has previously been shown to have the capacity to increase depolarization-evoked glutamate release in rat hippocampal nerve terminals. As cGMP-dependent signaling cascade has been found to modulate glutamate release at the presynaptic level, the aim of this study was to further examine the role of cGMP signaling pathway in the regulation of osthole on glutamate release in hippocampal synaptosomes. Results showed that osthole dose-dependently increased intrasynaptosomal cGMP levels. The elevation of cGMP levels by osthole was prevented by the phosphodiesterase 5 inhibitor sildenafil but was insensitive to the guanylyl cyclase inhibitor ODQ. In addition, osthole-induced facilitation of 4-aminopyridine (4-AP)-evoked glutamate release was completely prevented by the cGMP-dependent protein kinase (PKG) inhibitors, KT5823, and Rp-8-Br-PET-cGMPS. Direct activation of PKG with 8-Br-cGMP or 8-pCPT-cGMP also occluded the osthole-mediated facilitation of 4-AP-evoked glutamate release. Furthermore, sildenafil exhibited a dose-dependent facilitation of 4-AP-evoked release of glutamate and occluded the effect of osthole on the 4-AP-evoked glutamate release. Collectively, our findings suggest that osthole-mediated facilitation of glutamate release involves the activation of cGMP/PKG-dependent pathway. Copyright © 2011 Wiley Periodicals, Inc.

The Coexistence of Hypertension and Ovariectomy Additively Increases Cardiac Apoptosis.

PubMed

Lin, Yi-Yuan; Cheng, Yu-Jung; Hu, Jun; Chu, Li-Xi; Shyu, Woei-Cherng; Kao, Chung-Lan; Lin, Tzer-Bin; Kuo, Chia-Hua; Yang, Ai-Lun; Lee, Shin-Da

2016-12-06

To investigate whether the coexistence of hypertension and ovariectomy will increase cardiac Fas receptor and mitochondrial-dependent apoptotic pathways, histopathological analysis, the TUNEL assay and Western blotting were performed on the excised hearts from three groups of female spontaneously hypertensive rats (SHR), which were divided into a sham-operated group (SHR-Sham), bilaterally ovariectomized group (SHR-OVX) and normotensive Wistar Kyoto rats (WKY). Compared with the WKY group, the SHR-Sham group exhibited decreased protein levels of ERα, ERβ, p-Akt/Akt, Bcl-2, Bcl-xL and p-Bad and decreased further in the SHR-OVX group, as well as protein levels of t-Bid, Bak, Bad, Bax, cytochrome c , activated caspase-9 and activated caspase-3 (mitochondria-dependent apoptosis) increased in the SHR-Sham group and increased further in the SHR-OVX group. Compared with the WKY group, protein levels of Fas ligand, TNF-α, Fas death receptors, TNFR1, FADD and activated caspase-8 (Fas receptor-dependent apoptosis) increased in the SHR-Sham group, but did not increase in the SHR-OVX group, except Fas ligand and TNF-α. The coexistence of hypertension and ovariectomy attenuated the estrogen receptor survival pathway and appeared to additively increase the cardiac mitochondria-dependent, but not the Fas receptor-dependent apoptosis pathway, which might provide one possible mechanism for the development of cardiac abnormalities in hypertensive postmenopausal women.

Loss of CHK1 function impedes DNA damage-induced FANCD2 monoubiquitination but normalizes the abnormal G2 arrest in Fanconi anemia.

PubMed

Guervilly, Jean-Hugues; Macé-Aimé, Gaëtane; Rosselli, Filippo

2008-03-01

Fanconi anemia (FA) is a cancer-prone hereditary disease resulting from mutations in one of the 13 genes defining the FANC/BRCA pathway. This pathway is involved in the cellular resistance to DNA-cross-linking agents. How the FANC/BRCA pathway is activated and why its deficiency leads to the accumulation of FA cells with a 4N DNA content are still poorly answered questions. We investigated the involvement of ATR pathway members in these processes. We show here that RAD9 and RAD17 are required for DNA interstrand cross-link (ICL) resistance and for the optimal activation of FANCD2. Moreover, we demonstrate that CHK1 and its interacting partner CLASPIN that act downstream in the ATR pathway are required for both FANCD2 monoubiquitination and assembling in subnuclear foci in response to DNA damage. Paradoxically, in the absence of any genotoxic stress, CHK1 or CLASPIN depletion results in an increased basal level of FANCD2 monoubiquitination and focalization. We also demonstrate that the ICL-induced accumulation of FA cells in late S/G2 phase is dependent on ATR and CHK1. In agreement with this, CHK1 phosphorylation is enhanced in FA cells, and chemical inhibition of the ATR/CHK1 axis in FA lymphoblasts decreases their sensitivity to mitomycin C. In conclusion, this work describes a complex crosstalk between CHK1 and the FANC/BRCA pathway: CHK1 activates this pathway through FANCD2 monoubiquitination, whereas FA deficiency leads to a CHK1-dependent G2 accumulation, raising the possibility that the FANC/BRCA pathway downregulates CHK1 activation.

Nonylphenol regulates cyclooxygenase-2 expression via Ros-activated NF-κB pathway in sertoli TM4 cells.

PubMed

Liu, Xiaozhen; Nie, Shaoping; Huang, Danfei; Xie, Mingyong

2015-09-01

The aim of this study was to investigate the signaling pathways involved in the cyclooxygenase (COX)-2 regulation induced by nonylphenol (NP) in mouse testis Sertoli TM4 cells. Our results showed that treatment of TM4 cells with NP increased COX-2 protein expression and interleukin-6 (IL)-6 and prostaglandin E2 (PGE2) secretion in a dose-dependent manner. Pretreatment with reactive oxygen species (ROS) scavenger, N-acetylcysteine (NAC), attenuated NP-induced ROS production, COX-2 expression, and IL-6 and PGE2 release in TM4 cells. Exposure to NP stimulated activation of NF-κB, whereas the NF-κB inhibitor, pyrrolidine dithiocarbamate, attenuated NP-enhanced COX-2 expression and IL-6 and PGE2 release in TM4 cells in a dose-dependent manner. Furthermore, NAC blocked NP-induced activation of NF-κB. In addition, inhibition of COX-2 mitigated NP-induced IL-6 release. In conclusion, NP induced ROS generation, activation of NF-κB pathway, COX-2 upregulation, and IL-6 and PGE2 secretion in TM4 cells. NP may regulate COX-2 expression via ROS-activated NF-κB pathway in Sertoli TM4 cells. © 2014 Wiley Periodicals, Inc.

Endogenous enzyme activities and polyamine levels in diverse rice cultivars depend on the genetic background and are not affected by the presence of the hygromycin phosphotransferase selectable marker.

PubMed

Lepri, O.; Bassie, L.; Thu-Hang, P.; Christou, P.; Capell, T.

2002-09-01

We used the polyamine biosynthetic pathway and rice as a relevant model to understand the genetic basis of variation in endogenous levels of metabolites and key enzymes involved in the pathway. Wild-type tissues and also tissues containing a commonly used selectable marker gene were employed. We detected a wide variation in levels of arginine decarboxylase activity and in the three polyamines, putrescine, spermidine and spermine, in different tissues and varieties, but this was not dependent on the presence of the selectable marker. A more-extensive profile of enzyme activities (ADC, ODC, SAMDC, DAO and PAO) and polyamine levels in different tissues was generated in two different varieties. Our results indicate that genetic background is important in terms of the basal levels of metabolites and enzyme activity, particularly in situations in which we aim to engineer metabolic pathways that are also encoded by homologous endogenous genes. We did not find any evidence that the presence of a selectable marker in any way influences enzyme activity or metabolite levels.

The RhoA-ROCK-PTEN pathway as a molecular switch for anchorage dependent cell behavior.

PubMed

Yang, Seungwon; Kim, Hyun-Man

2012-04-01

The proliferation of anchorage-dependent cells of mesenchymal origin requires the attachment of the cells to substrates. Thus, cells that are poorly attached to substrates exhibit retarded cell cycle progression or apoptotic death. A major disadvantage of most polymers used in tissue engineering is their hydrophobicity; hydrophobic surfaces do not allow cells to attach firmly and, therefore, do not allow normal proliferation rates. In this study, we investigated the molecular mechanism underlying the reduced proliferation rate of cells that are poorly attached to substrates. There was an inverse relationship between the activity of the small GTPase RhoA (RhoA) and the cell proliferation rate. RhoA activity correlated inversely with the strength of cell adhesion to the substrates. The high RhoA activity in the cells poorly attached to substrates caused an increase in the activity of Rho-associated kinase (ROCK), a well-known effector of RhoA that upregulated the activity of phosphatase and tensin homolog (PTEN). The resulting activated PTEN downregulated Akt activity, which is essential for cell proliferation. Thus, the cells that were poorly attached to substrates showed low levels of cell proliferation because the RhoA-ROCK-PTEN pathway was hyperactive. In addition, RhoA activity seemed to be related to focal adhesion kinase (FAK) activity. Weak FAK activity in these poorly attached cells failed to downregulate the high RhoA activity that restrained cell proliferation. Interestingly, reducing the expression of any component of the RhoA-ROCK-PTEN pathway rescued the proliferation rate without physico-chemical surface modifications. Based on these results, we suggest that the RhoA-ROCK-PTEN pathway acts as a molecular switch to control cell proliferation and determine anchorage dependence. In cells that are poorly attached to substrates, its inhibition is sufficient to restore cell proliferation without the need for physico-chemical modification of the material surface. Copyright © 2012 Elsevier Ltd. All rights reserved.

GAS6-expressing and self-sustaining cancer cells in 3D spheroids activate the PDK-RSK-mTOR pathway for survival and drug resistance.

PubMed

Baumann, Christine; Ullrich, Axel; Torka, Robert

2017-10-01

AXL receptor tyrosine kinase (RTK) inhibition presents a promising therapeutic strategy for aggressive tumor subtypes, as AXL signaling is upregulated in many cancers resistant to first-line treatments. Furthermore, the AXL ligand growth arrest-specific gene 6 (GAS6) has recently been linked to cancer drug resistance. Here, we established that challenging conditions, such as serum deprivation, divide AXL-overexpressing tumor cell lines into non-self-sustaining and self-sustaining subtypes in 3D spheroid culture. Self-sustaining cells are characterized by excessive GAS6 secretion and TAM-PDK-RSK-mTOR pathway activation. In 3D spheroid culture, the activation of the TAM-PDK-RSK-mTOR pathway proves crucial following treatment with AXL/MET inhibitor BMS777607, when the self-sustaining tumor cells react with TAM-RSK hyperactivation and enhanced SRC-AKT-mTOR signaling. Thus, bidirectional activated mTOR leads to enhanced proliferation and counteracts the drug effect. mTOR activation is accompanied by an enhanced AXL expression and hyperphosphorylation following 24 h of treatment with BMS777607. Therefore, we elucidate a double role of AXL that can be assigned to RSK-mTOR as well as SRC-AKT-mTOR pathway activation, specifically through AXL Y779 phosphorylation. This phosphosite fuels the resistance mechanism in 3D spheroids, alongside further SRC-dependent EGFR Y1173 and/or MET Y1349 phosphorylation which is defined by the cell-specific addiction. In conclusion, self-sustenance in cancer cells is based on a signaling synergy, individually balanced between GAS6 TAM-dependent PDK-RSK-mTOR survival pathway and the AXLY779/EGFR/MET-driven SRC-mTOR pathway. © 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

Concurrent activation of striatal direct and indirect pathways during action initiation.

PubMed

Cui, Guohong; Jun, Sang Beom; Jin, Xin; Pham, Michael D; Vogel, Steven S; Lovinger, David M; Costa, Rui M

2013-02-14

The basal ganglia are subcortical nuclei that control voluntary actions, and they are affected by a number of debilitating neurological disorders. The prevailing model of basal ganglia function proposes that two orthogonal projection circuits originating from distinct populations of spiny projection neurons (SPNs) in the striatum--the so-called direct and indirect pathways--have opposing effects on movement: activity of direct-pathway SPNs is thought to facilitate movement, whereas activity of indirect-pathway SPNs is presumed to inhibit movement. This model has been difficult to test owing to the lack of methods to selectively measure the activity of direct- and indirect-pathway SPNs in freely moving animals. Here we develop a novel in vivo method to specifically measure direct- and indirect-pathway SPN activity, using Cre-dependent viral expression of the genetically encoded calcium indicator (GECI) GCaMP3 in the dorsal striatum of D1-Cre (direct-pathway-specific) and A2A-Cre (indirect-pathway-specific) mice. Using fibre optics and time-correlated single-photon counting (TCSPC) in mice performing an operant task, we observed transient increases in neural activity in both direct- and indirect-pathway SPNs when animals initiated actions, but not when they were inactive. Concurrent activation of SPNs from both pathways in one hemisphere preceded the initiation of contraversive movements and predicted the occurrence of specific movements within 500 ms. These observations challenge the classical view of basal ganglia function and may have implications for understanding the origin of motor symptoms in basal ganglia disorders.

Aripiprazole and Haloperidol Activate GSK3β-Dependent Signalling Pathway Differentially in Various Brain Regions of Rats.

PubMed

Pan, Bo; Huang, Xu-Feng; Deng, Chao

2016-03-28

Aripiprazole, a dopamine D₂ receptor (D₂R) partial agonist, possesses a unique clinical profile. Glycogen synthase kinase 3β (GSK3β)-dependent signalling pathways have been implicated in the pathophysiology of schizophrenia and antipsychotic drug actions. The present study examined whether aripiprazole differentially affects the GSK3β-dependent signalling pathways in the prefrontal cortex (PFC), nucleus accumbens (NAc), and caudate putamen (CPu), in comparison with haloperidol (a D₂R antagonist) and bifeprunox (a D₂R partial agonist). Rats were orally administrated aripiprazole (0.75 mg/kg), bifeprunox (0.8 mg/kg), haloperidol (0.1 mg/kg) or vehicle three times per day for one week. The levels of protein kinase B (Akt), p-Akt, GSK3β, p-GSK3β, dishevelled (Dvl)-3, and β-catenin were measured by Western Blots. Aripiprazole increased GSK3β phosphorylation in the PFC and NAc, respectively, while haloperidol elevated it in the NAc only. However, Akt activity was not changed by any of these drugs. Additionally, both aripiprazole and haloperidol, but not bifeprunox, increased the expression of Dvl-3 and β-catenin in the NAc. The present study suggests that activation of GSK3β phosphorylation in the PFC and NAc may be involved in the clinical profile of aripiprazole; additionally, aripiprazole can increase GSK3β phosphorylation via the Dvl-GSK3β-β-catenin signalling pathway in the NAc, probably due to its relatively low intrinsic activity at D₂Rs.

Angiotensin II promotes the proliferation of activated pancreatic stellate cells by Smad7 induction through a protein kinase C pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hama, Kouji; Ohnishi, Hirohide; Aoki, Hiroyoshi

2006-02-17

Activated pancreatic stellate cells (PSCs) play major roles in promoting pancreatic fibrosis. We previously reported that angiotensin II (Ang II) enhances activated PSC proliferation through EGF receptor transactivation. In the present study, we elucidated a novel intracellular mechanism by which Ang II stimulates cellular proliferation. TGF-{beta}{sub 1} inhibits activated PSC proliferation via a Smad3 and Smad4-dependent pathway in an autocrine manner. We demonstrated that Ang II inhibited TGF-{beta}{sub 1}-induced nuclear accumulation of Smad3 and Smad4. Furthermore, Ang II rapidly induced inhibitory Smad7 mRNA expression. Adenovirus-mediated Smad7 overexpression inhibited TGF-{beta}{sub 1}-induced nuclear accumulation of Smad3 and Smad4, and potentiated activated PSCmore » proliferation. PKC inhibitor Go6983 blocked the induction of Smad7 mRNA expression by Ang II. In addition, 12-O-tetradecanoyl-phorbol 13-acetate, a PKC activator, increased Smad7 mRNA expression. These results suggest that Ang II enhances activated PSC proliferation by blocking autocrine TGF-{beta}{sub 1}-mediated growth inhibition by inducing Smad7 expression via a PKC-dependent pathway.« less

Tryptophan depletion under conditions that imitate insulin resistance enhances fatty acid oxidation and induces endothelial dysfunction through reactive oxygen species-dependent and independent pathways.

PubMed

Eleftheriadis, Theodoros; Pissas, Georgios; Sounidaki, Maria; Antoniadi, Georgia; Rountas, Christos; Liakopoulos, Vassilios; Stefanidis, Loannis

2017-04-01

In atherosclerosis-associated pathologic entities characterized by malnutrition and inflammation, L-tryptophan (TRP) levels are low. Insulin resistance is an independent cardiovascular risk factor and induces endothelial dysfunction by increasing fatty acid oxidation. It is also associated with inflammation and low TRP levels. Low TRP levels have been related to worse cardiovascular outcome. This study evaluated the effect of TRP depletion on endothelial dysfunction under conditions that imitate insulin resistance. Fatty acid oxidation, harmful pathways due to increased fatty acid oxidation, and endothelial dysfunction were assessed in primary human aortic endothelial cells cultured under normal glucose, low insulin conditions in the presence or absence of TRP. TRP depletion activated general control non-derepressible 2 kinase and inhibited aryl hydrocarbon receptor. It increased fatty acid oxidation by increasing expression and activity of carnitine palmitoyltransferase 1. Elevated fatty acid oxidation increased the formation of reactive oxygen species (ROS) triggering the polyol and hexosamine pathways, and enhancing protein kinase C activity and methylglyoxal production. TRP absence inhibited nitric oxide synthase activity in a ROS-dependent way, whereas it increased the expression of ICAM-1 and VCAM-1 in a ROS independent and possibly p53-dependent manner. Thus, TRP depletion, an amino acid whose low levels have been related to worse cardiovascular outcome and to inflammatory atherosclerosis-associated pathologic entities, under conditions that imitate insulin resistance enhances fatty acid oxidation and induces endothelial dysfunction through ROS-dependent and independent pathways. These findings may offer new insights at the molecular mechanisms involved in accelerated atherosclerosis that frequently accompanies malnutrition and inflammation.

Acentriolar mitosis activates a p53-dependent apoptosis pathway in the mouse embryo

PubMed Central

Bazzi, Hisham; Anderson, Kathryn V.

2014-01-01

Centrosomes are the microtubule-organizing centers of animal cells that organize interphase microtubules and mitotic spindles. Centrioles are the microtubule-based structures that organize centrosomes, and a defined set of proteins, including spindle assembly defective-4 (SAS4) (CPAP/CENPJ), is required for centriole biogenesis. The biological functions of centrioles and centrosomes vary among animals, and the functions of mammalian centrosomes have not been genetically defined. Here we use a null mutation in mouse Sas4 to define the cellular and developmental functions of mammalian centrioles in vivo. Sas4-null embryos lack centrosomes but survive until midgestation. As expected, Sas4−/− mutants lack primary cilia and therefore cannot respond to Hedgehog signals, but other developmental signaling pathways are normal in the mutants. Unlike mutants that lack cilia, Sas4−/− embryos show widespread apoptosis associated with global elevated expression of p53. Cell death is rescued in Sas4−/− p53−/− double-mutant embryos, demonstrating that mammalian centrioles prevent activation of a p53-dependent apoptotic pathway. Expression of p53 is not activated by abnormalities in bipolar spindle organization, chromosome segregation, cell-cycle profile, or DNA damage response, which are normal in Sas4−/− mutants. Instead, live imaging shows that the duration of prometaphase is prolonged in the mutants while two acentriolar spindle poles are assembled. Independent experiments show that prolonging spindle assembly is sufficient to trigger p53-dependent apoptosis. We conclude that a short delay in the prometaphase caused by the absence of centrioles activates a previously undescribed p53-dependent cell death pathway in the rapidly dividing cells of the mouse embryo. PMID:24706806

Fenofibrate activates Nrf2 through p62-dependent Keap1 degradation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Jeong Su; Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752; Kang, Dong Hoon

Peroxisome proliferator-activated receptor α (PPARα) activates the β-oxidation of fatty acids in the liver. Fenofibrate is a potent agonist of PPARα and is used in the treatment of hyperlipidemia. Fenofibrate treatment often induces the production of intracellular reactive oxygen species (ROS), leading to cell death. The nuclear factor erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway is an essential component of the defense mechanism against oxidative stress. However, the molecular mechanism underlying the regulation of the Nrf2-Keap1 pathway in fenofibrate-induced cell death is not known. In this study, we demonstrated that fenofibrate induces Keap1 degradation and Nrf2 activation.more » This fenofibrate-mediated Keap1 degradation is partly dependent on autophagy. Furthermore, fenofibrate-induced Keap1 degradation followed by Nrf2 activation is mainly mediated by p62, which functions as an adaptor protein in the autophagic pathway. Consistent with these findings, ablation of p62 increased fenofibrate-mediated apoptotic cell death associated with ROS accumulation. These results strongly suggest that p62 plays a crucial role in preventing fenofibrate-induced cell death. - Highlights: • Fenofibrate induces cell death by increasing ROS production. • The underlying defense mechanism against this effect is unknown. • Fenofibrate induces autophagy-dependent Keap1 degradation and Nrf2 activation. • This process is p62-dependent; lack of p62 enhanced fenofibrate-mediated apoptosis. • p62 plays a crucial role in preventing fenofibrate-induced cell death.« less

InsR/IGF1R pathway mediates resistance to EGFR inhibitors in glioblastoma

PubMed Central

Ma, Yufang; Tang, Nan; Thompson, Reid; Mobley, Bret C.; Clark, Steven W.; Sarkaria, Jann N.; Wang, Jialiang

2015-01-01

Purpose Aberrant activation of epidermal growth factor receptor (EGFR) is a hallmark of glioblastoma. However, EGFR inhibitors exhibit at best modest efficacy in glioblastoma. This is in sharp contrast to the observations in EGFR-mutant lung cancer. We examined whether activation of functionally redundant receptor tyrosine kinases (RTKs) conferred resistance to EGFR inhibitors in glioblastoma. Experimental Design We collected a panel of patient-derived glioblastoma xenograft (PDX) lines that maintained expression of wild type or mutant EGFR in serial xenotransplantation and tissue cultures. Using this physiologically relevant platform, we tested the abilities of several RTK ligands to protect glioblastoma cells against an EGFR inhibitor, gefitinib. Based on the screening results, we further developed a combination therapy co-targeting EGFR and insulin receptor (InsR)/insulin-like growth factor 1 receptor (IGF1R). Results Insulin and IGF1 induced significant protection against gefitinib in the majority of EGFR-dependent PDX lines with one exception that did not expression InsR or IGF1R. Blockade of the InsR/IGF1R pathway synergistically improved sensitivity to gefitinib or dacomitinib. Gefitinib alone effectively attenuated EGFR activities and the downstream MEK/ERK pathway. However, repression of AKT and induction of apoptosis required concurrent inhibition of both EGFR and InsR/IGF1R. A combination of gefitinib and OSI-906, a dual InsR/IGF1R inhibitor, was more effective than either agent alone to treat subcutaneous glioblastoma xenograft tumors. Conclusions Our results suggest that activation of the InsR/IGF1R pathway confers resistance to EGFR inhibitors in EGFR-dependent glioblastoma through AKT regulation. Concurrent blockade of these two pathways holds promise to treat EGFR-dependent glioblastoma. PMID:26561558

Hydrogen-rich medium protects mouse embryonic fibroblasts from oxidative stress by activating LKB1-AMPK-FoxO1 signal pathway.

PubMed

Lee, Jihyun; Yang, Goowon; Kim, Young-Joo; Tran, Quynh Hoa; Choe, Wonchae; Kang, Insug; Kim, Sung Soo; Ha, Joohun

2017-09-23

Persistent oxidative stress is recognized as a major cause of many pathological conditions as well as ageing. However, most clinical trials of dietary antioxidants have failed to produce successful outcomes in treating oxidative stress-induced diseases. Molecular hydrogen (H 2 ) has recently received considerable attention as a therapeutic agent owing to its novel antioxidant properties, a selective scavenger of hydroxyl and peroxynitrite radicals. Beyond this, numerous reports support that H 2 can modulate the activity of various cellular signal pathways. However, its effect on AMP-activated protein kinase (AMPK) signal pathway, a central regulator of energy hemostasis, has remained almost elusive. Here, we report that hydrogen-rich medium activated LKB1-AMPK signal pathway without ATP depletion, which in turn induced FoxO1-dependent transcription of manganese superoxide dismutase and catalase in mouse embryonic fibroblasts. Moreover, hydrogen-rich media effectively reduced the level of reactive oxygen species in cells treated with hydrogen peroxide and protected these cells from apoptosis in an AMPK-dependent manner. These results suggest that the LKB1-AMPK-FoxO1 signaling pathway is a critical mediator of the antioxidant properties of H 2 , further supporting the idea that H 2 acts as a signaling molecule to serve various physiological functions. Copyright © 2017 Elsevier Inc. All rights reserved.

Anti-neuroinflammatory efficacy of the aldose reductase inhibitor FMHM via phospholipase C/protein kinase C-dependent NF-κB and MAPK pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zeng, Ke-Wu; Li, Jun; Dong, Xin

2013-11-15

Aldose reductase (AR) has a key role in several inflammatory diseases: diabetes, cancer and cardiovascular diseases. Therefore, AR inhibition seems to be a useful strategy for anti-inflammation therapy. In the central nervous system (CNS), microglial over-activation is considered to be a central event in neuroinflammation. However, the effects of AR inhibition in CNS inflammation and its underlying mechanism of action remain unknown. In the present study, we found that FMHM (a naturally derived AR inhibitor from the roots of Polygala tricornis Gagnep.) showed potent anti-neuroinflammatory effects in vivo and in vitro by inhibiting microglial activation and expression of inflammatory mediators.more » Mechanistic studies showed that FMHM suppressed the activity of AR-dependent phospholipase C/protein kinase C signaling, which further resulted in downstream inactivation of the IκB kinase/IκB/nuclear factor-kappa B (NF-κB) inflammatory pathway. Therefore, AR inhibition-dependent NF-κB inactivation negatively regulated the transcription and expression of various inflammatory genes. AR inhibition by FMHM exerted neuroprotective effects in lipopolysaccharide-induced neuron–microglia co-cultures. These findings suggested that AR is a potential target for neuroinflammation inhibition and that FMHM could be an effective agent for treating or preventing neuroinflammatory diseases. - Highlights: • FMHM is a natural-derived aldose reductase (AR) inhibitor. • FMHM inhibits various neuroinflammatory mediator productions in vitro and in vivo. • FMHM inhibits neuroinflammation via aldose reductase/PLC/PKC-dependent NF-κB pathway. • FMHM inhibits neuroinflammation via aldose reductase/PLC/PKC-dependent MAPK pathway. • FMHM protects neurons against inflammatory injury in microglia-neuron co-cultures.« less

Neuronal Ca2+ sensor-1 contributes to stress tolerance in cardiomyocytes via activation of mitochondrial detoxification pathways.

PubMed

Nakamura, Tomoe Y; Nakao, Shu; Wakabayashi, Shigeo

2016-10-01

Identification of the molecules involved in cell death/survival pathways is important for understanding the mechanisms of cell loss in cardiac disease, and thus is clinically relevant. Ca 2+ -dependent signals are often involved in these pathways. Here, we found that neuronal Ca 2+ -sensor-1 (NCS-1), a Ca 2+ -binding protein, has an important role in cardiac survival during stress. Cardiomyocytes derived from NCS-1-deficient (Ncs1 -/- ) mice were more susceptible to oxidative and metabolic stress than wild-type (WT) myocytes. Cellular ATP levels and mitochondrial respiration rates, as well as the levels of mitochondrial marker proteins, were lower in Ncs1 -/- myocytes. Although oxidative stress elevated mitochondrial proton leak, which exerts a protective effect by inhibiting the production of reactive oxygen species in WT myocytes, this response was considerably diminished in Ncs1 -/- cardiomyocytes, and this would be a major reason for cell death. Consistently, H 2 O 2 -induced loss of mitochondrial membrane potential, a critical early event in cell death, was accelerated in Ncs1 -/- myocytes. Furthermore, NCS-1 was upregulated in hearts subjected to ischemia-reperfusion, and ischemia-reperfusion injury was more severe in Ncs1 -/- hearts. Activation of stress-induced Ca 2+ -dependent survival pathways, such as Akt and PGC-1α (which promotes mitochondrial biogenesis and function), was diminished in Ncs1 -/- hearts. Overall, these data demonstrate that NCS-1 contributes to stress tolerance in cardiomyocytes at least in part by activating certain Ca 2+ -dependent survival pathways that promote mitochondrial biosynthesis/function and detoxification pathways. Copyright © 2016 Elsevier Ltd. All rights reserved.

Phosphorylation of PPP(S/T)P motif of the free LRP6 intracellular domain is not required to activate the Wnt/beta-catenin pathway and attenuate GSK3beta activity.

PubMed

Beagle, Brandon; Mi, Kaihong; Johnson, Gail V W

2009-11-01

The canonical Wnt/beta-catenin signaling pathway plays a critical role in numerous physiological and pathological processes. LRP6 is an essential co-receptor for Wnt/beta-catenin signaling; as transduction of the Wnt signal is strongly dependent upon GSK3beta-mediated phosphorylation of multiple PPP(S/T)P motifs within the membrane-anchored LRP6 intracellular domain. Previously, we showed that the free LRP6 intracellular domain (LRP6-ICD) can activate the Wnt/beta-catenin pathway in a beta-catenin and TCF/LEF-1 dependent manner, as well as interact with and attenuate GSK3beta activity. However, it is unknown if the ability of LRP6-ICD to attenuate GSK3beta activity and modulate activation of the Wnt/beta-catenin pathway requires phosphorylation of the LRP6-ICD PPP(S/T)P motifs, in a manner similar to the membrane-anchored LRP6 intracellular domain. Here we provide evidence that the LRP6-ICD does not have to be phosphorylated at its PPP(S/T)P motif by GSK3beta to stabilize endogenous cytosolic beta-catenin resulting in activation of TCF/LEF-1 and the Wnt/beta-catenin pathway. LRP6-ICD and a mutant in which all 5 PPP(S/T)P motifs were changed to PPP(A)P motifs equivalently interacted with and attenuated GSK3beta activity in vitro, and both constructs inhibited the in situ GSK3beta-mediated phosphorylation of beta-catenin and tau to the same extent. These data indicate that the LRP6-ICD attenuates GSK3beta activity similar to other GSK3beta binding proteins, and is not a result of it being a GSK3beta substrate. Our findings suggest the functional and regulatory mechanisms governing the free LRP6-ICD may be distinct from membrane-anchored LRP6, and that release of the LRP6-ICD may provide a complimentary signaling cascade capable of modulating Wnt-dependent gene expression. (c) 2009 Wiley-Liss, Inc.

AMP-activated protein kinase-mediated feedback phosphorylation controls the Ca2+/calmodulin (CaM) dependence of Ca2+/CaM-dependent protein kinase kinase β.

PubMed

Nakanishi, Akihiro; Hatano, Naoya; Fujiwara, Yuya; Sha'ri, Arian; Takabatake, Shota; Akano, Hiroki; Kanayama, Naoki; Magari, Masaki; Nozaki, Naohito; Tokumitsu, Hiroshi

2017-12-01

The Ca 2+ /calmodulin-dependent protein kinase kinase β (CaMKKβ)/5'-AMP-activated protein kinase (AMPK) phosphorylation cascade affects various Ca 2+ -dependent metabolic pathways and cancer growth. Unlike recombinant CaMKKβ that exhibits higher basal activity (autonomous activity), activation of the CaMKKβ/AMPK signaling pathway requires increased intracellular Ca 2+ concentrations. Moreover, the Ca 2+ /CaM dependence of CaMKKβ appears to arise from multiple phosphorylation events, including autophosphorylation and activities furnished by other protein kinases. However, the effects of proximal downstream kinases on CaMKKβ activity have not yet been evaluated. Here, we demonstrate feedback phosphorylation of CaMKKβ at multiple residues by CaMKKβ-activated AMPK in addition to autophosphorylation in vitro , leading to reduced autonomous, but not Ca 2+ /CaM-activated, CaMKKβ activity. MS analysis and site-directed mutagenesis of AMPK phosphorylation sites in CaMKKβ indicated that Thr 144 phosphorylation by activated AMPK converts CaMKKβ into a Ca 2+ /CaM-dependent enzyme as shown by completely Ca 2+ /CaM-dependent CaMKK activity of a phosphomimetic T144E CaMKKβ mutant. CaMKKβ mutant analysis indicated that the C-terminal domain (residues 471-587), including the autoinhibitory region, plays an important role in stabilizing an inactive conformation in a Thr 144 phosphorylation-dependent manner. Furthermore, immunoblot analysis with anti-phospho-Thr 144 antibody revealed phosphorylation of Thr 144 in CaMKKβ in transfected COS-7 cells that was further enhanced by exogenous expression of AMPKα. These results indicate that AMPK-mediated feedback phosphorylation of CaMKKβ regulates the CaMKKβ/AMPK signaling cascade and may be physiologically important for intracellular maintenance of Ca 2+ -dependent AMPK activation by CaMKKβ. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

New Insights into the Compositional Dependence of Li-Ion Transport in Polymer-Ceramic Composite Electrolytes.

PubMed

Zheng, Jin; Hu, Yan-Yan

2018-01-31

Composite electrolytes are widely studied for their potential in realizing improved ionic conductivity and electrochemical stability. Understanding the complex mechanisms of ion transport within composites is critical for effectively designing high-performance solid electrolytes. This study examines the compositional dependence of the three determining factors for ionic conductivity, including ion mobility, ion transport pathways, and active ion concentration. The results show that with increase in the fraction of ceramic Li 7 La 3 Zr 2 O 12 (LLZO) phase in the LLZO-poly(ethylene oxide) composites, ion mobility decreases, ion transport pathways transit from polymer to ceramic routes, and the active ion concentration increases. These changes in ion mobility, transport pathways, and concentration collectively explain the observed trend of ionic conductivity in composite electrolytes. Liquid additives alter ion transport pathways and increase ion mobility, thus enhancing ionic conductivity significantly. It is also found that a higher content of LLZO leads to improved electrochemical stability of composite electrolytes. This study provides insight into the recurring observations of compositional dependence of ionic conductivity in current composite electrolytes and pinpoints the intrinsic limitations of composite electrolytes in achieving fast ion conduction.

Class I ADP-Ribosylation Factors Are Involved in Enterovirus 71 Replication

PubMed Central

Wang, Jianmin; Du, Jiang; Jin, Qi

2014-01-01

Enterovirus 71 is one of the major causative agents of hand, foot, and mouth disease in infants and children. Replication of enterovirus 71 depends on host cellular factors. The viral replication complex is formed in novel, cytoplasmic, vesicular compartments. It has not been elucidated which cellular pathways are hijacked by the virus to create these vesicles. Here, we investigated whether proteins associated with the cellular secretory pathway were involved in enterovirus 71 replication. We used a loss-of-function assay, based on small interfering RNA. We showed that enterovirus 71 RNA replication was dependent on the activity of Class I ADP-ribosylation factors. Simultaneous depletion of ADP-ribosylation factors 1 and 3, but not three others, inhibited viral replication in cells. We also demonstrated with various techniques that the brefeldin-A-sensitive guanidine nucleotide exchange factor, GBF1, was critically important for enterovirus 71 replication. Our results suggested that enterovirus 71 replication depended on GBF1-mediated activation of Class I ADP-ribosylation factors. These results revealed a connection between enterovirus 71 replication and the cellular secretory pathway; this pathway may represent a novel target for antiviral therapies. PMID:24911624

NF-κB signaling pathways: role in nervous system physiology and pathology.

PubMed

Mincheva-Tasheva, Stefka; Soler, Rosa M

2013-04-01

Intracellular pathways related to cell survival regulate neuronal physiology during development and neurodegenerative disorders. One of the pathways that have recently emerged with an important role in these processes is nuclear factor-κB (NF-κB). The activity of this pathway leads to the nuclear translocation of the NF-κB transcription factors and the regulation of anti-apoptotic gene expression. Different stimuli can activate the pathway through different intracellular cascades (canonical, non-canonical, and atypical), contributing to the translocation of specific dimers of the NF-κB transcription factors, and each of these dimers can regulate the transcription of different genes. Recent studies have shown that the activation of this pathway regulates opposite responses such as cell survival or neuronal degeneration. These apparent contradictory effects depend on conditions such as the pathway stimuli, the origin of the cells, or the cellular context. In the present review, the authors summarize these findings and discuss their significance with respect to survival or death in the nervous system.

Nitric oxide/cGMP pathway signaling actively down-regulates α4β1-integrin affinity: an unexpected mechanism for inducing cell de-adhesion.

PubMed

Chigaev, Alexandre; Smagley, Yelena; Sklar, Larry A

2011-05-17

Integrin activation in response to inside-out signaling serves as the basis for rapid leukocyte arrest on endothelium, migration, and mobilization of immune cells. Integrin-dependent adhesion is controlled by the conformational state of the molecule, which is regulated by seven-transmembrane Guanine nucleotide binding Protein-Coupled Receptors (GPCRs). α4β1-integrin (CD49d/CD29, Very Late Antigen-4, VLA-4) is expressed on leukocytes, hematopoietic progenitors, stem cells, hematopoietic cancer cells, and others. VLA-4 conformation is rapidly up-regulated by inside-out signaling through Gαi-coupled GPCRs and down-regulated by Gαs-coupled GPCRs. However, other signaling pathways, which include nitric oxide-dependent signaling, have been implicated in the regulation of cell adhesion. The goal of the current report was to study the effect of nitric oxide/cGMP signaling pathway on VLA-4 conformational regulation. Using fluorescent ligand binding to evaluate the integrin activation state on live cells in real-time, we show that several small molecules, which specifically modulate nitric oxide/cGMP signaling pathway, as well as a cell permeable cGMP analog, can rapidly down-modulate binding of a VLA-4 specific ligand on cells pre-activated through three Gαi-coupled receptors: wild type CXCR4, CXCR2 (IL-8RB), and a non-desensitizing mutant of formyl peptide receptor (FPR ΔST). Upon signaling, we detected rapid changes in the ligand dissociation rate. The dissociation rate after inside-out integrin de-activation was similar to the rate for resting cells. In a VLA-4/VCAM-1-specific myeloid cell adhesion system, inhibition of the VLA-4 affinity change by nitric oxide had a statistically significant effect on real-time cell aggregation. We conclude that nitric oxide/cGMP signaling pathway can rapidly down-modulate the affinity state of the VLA-4 binding pocket, especially under the condition of sustained Gαi-coupled GPCR signaling, generated by a non-desensitizing receptor mutant. This suggests a fundamental role of this pathway in de-activation of integrin-dependent cell adhesion.

Signaling pathways targeted by curcumin in acute and chronic injury: burns and photo-damaged skin.

PubMed

Heng, Madalene C Y

2013-05-01

Phosphorylase kinase (PhK) is a unique enzyme in which the spatial arrangements of the specificity determinants can be manipulated to allow the enzyme to recognize substrates of different specificities. In this way, PhK is capable of transferring high energy phosphate bonds from ATP to serine/threonine and tyrosine moieties in serine/threonine kinases and tyrosine kinases, thus playing a key role in the activation of multiple signaling pathways. Phosphorylase kinase is released within five minutes following injury and is responsible for activating inflammatory pathways in injury-activated scarring following burns. In photo-damaged skin, PhK plays an important role in promoting photocarcinogenesis through activation of NF-kB-dependent signaling pathways with inhibition of apoptosis of photo-damaged cells, thus promoting the survival of precancerous cells and allowing for subsequent tumor transformation. Curcumin, the active ingredient in the spice, turmeric, is a selective and non-competitive PhK inhibitor. By inhibition of PhK, curcumin targets multiple PhK-dependent pathways, with salutary effects on a number of skin diseases induced by injury. In this paper, we show that curcumin gel produces rapid healing of burns, with little or no residual scarring. Curcumin gel is also beneficial in the repair of photo-damaged skin, including pigmentary changes, solar elastosis, thinning of the skin with telangiectasia (actinic poikiloderma), and premalignant lesions such as actinic keratoses, dysplastic nevi, and advanced solar lentigines, but the repair process takes many months. © 2012 The International Society of Dermatology.

Endothelial NOS-dependent activation of c-Jun NH(2)- terminal kinase by oxidized low-density lipoprotein

NASA Technical Reports Server (NTRS)

Go, Y. M.; Levonen, A. L.; Moellering, D.; Ramachandran, A.; Patel, R. P.; Jo, H.; Darley-Usmar, V. M.

2001-01-01

Oxidized low-density lipoprotein (oxLDL) is known to activate a number of signal transduction pathways in endothelial cells. Among these are the c-Jun NH(2)-terminal kinase (JNK), also known as stress-activated protein kinase, and extracellular signal-regulated kinase (ERK). These mitogen-activated protein kinases (MAP kinase) determine cell survival in response to environmental stress. Interestingly, JNK signaling involves redox-sensitive mechanisms and is activated by reactive oxygen and nitrogen species derived from both NADPH oxidases, nitric oxide synthases (NOS), peroxides, and oxidized low-density lipoprotein (oxLDL). The role of endothelial NOS (eNOS) in the activation of JNK in response to oxLDL has not been examined. Herein, we show that on exposure of endothelial cells to oxLDL, both ERK and JNK are activated through independent signal transduction pathways. A key role of eNOS activation through a phosphatidylinositol-3-kinase-dependent mechanism leading to phosphorylation of eNOS is demonstrated for oxLDL-dependent activation of JNK. Moreover, we show that activation of ERK by oxLDL is critical in protection against the cytotoxicity of oxLDL.

The calcineurin pathway links hyperpolarization (Kir2.1)-induced Ca2+ signals to human myoblast differentiation and fusion.

PubMed

Konig, Stéphane; Béguet, Anne; Bader, Charles R; Bernheim, Laurent

2006-08-01

In human myoblasts triggered to differentiate, a hyperpolarization, resulting from K+ channel (Kir2.1) activation, allows the generation of an intracellular Ca2+ signal. This signal induces an increase in expression/activity of two key transcription factors of the differentiation process, myogenin and MEF2. Blocking hyperpolarization inhibits myoblast differentiation. The link between hyperpolarization-induced Ca2+ signals and the four main regulatory pathways involved in myoblast differentiation was the object of this study. Of the calcineurin, p38-MAPK, PI3K and CaMK pathways, only the calcineurin pathway was inhibited when Kir2.1-linked hyperpolarization was blocked. The CaMK pathway, although Ca2+ dependent, is unaffected by changes in membrane potential or block of Kir2.1 channels. Concerning the p38-MAPK and PI3K pathways, their activity is present already in proliferating myoblasts and they are unaffected by hyperpolarization or Kir2.1 channel block. We conclude that the Kir2.1-induced hyperpolarization triggers human myoblast differentiation via the activation of the calcineurin pathway, which, in turn, induces expression/activity of myogenin and MEF2.

Modulation of the Akt Pathway Reveals a Novel Link with PERK/eIF2α, which Is Relevant during Hypoxia

PubMed Central

Sánchez, Manuel Alejandro; Urrutia, Carolina; Grande, Alicia; Risso, Guillermo; Srebrow, Anabella; Alfaro, Jennifer; Colman-Lerner, Alejandro

2013-01-01

The unfolded protein response (UPR) and the Akt signaling pathway share several regulatory functions and have the capacity to determine cell outcome under specific conditions. However, both pathways have largely been studied independently. Here, we asked whether the Akt pathway regulates the UPR. To this end, we used a series of chemical compounds that modulate PI3K/Akt pathway and monitored the activity of the three UPR branches: PERK, IRE1 and ATF6. The antiproliferative and antiviral drug Akt-IV strongly and persistently activated all three branches of the UPR. We present evidence that activation of PERK/eIF2α requires Akt and that PERK is a direct Akt target. Chemical activation of this novel Akt/PERK pathway by Akt-IV leads to cell death, which was largely dependent on the presence of PERK and IRE1. Finally, we show that hypoxia-induced activation of eIF2α requires Akt, providing a physiologically relevant condition for the interaction between Akt and the PERK branch of the UPR. These data suggest the UPR and the Akt pathway signal to one another as a means of controlling cell fate. PMID:23922774

Cutting the brakes and flooring the gas: how TMEPAI turns TGF-β into a tumor promoter.

PubMed

Cichon, Magdalena A; Radisky, Derek C

2014-09-01

In normal or nonmalignant cells, TGF-β inhibits cellular proliferation through activation of the SMAD-dependent canonical signaling pathway. Recent findings demonstrate that the protein TMEPAI1 can block the cytostatic effects of the canonical TGF-β signaling pathway, while activating cellular proliferation through the noncanonical, SMAD-independent TGF-β signaling pathway. As TMEPAI1 shows increased expression in the poor prognosis basal and HER2 intrinsic subtypes of breast cancer, these findings point to a new avenue of targeted therapy with considerable therapeutic potential.

Transcriptional Pathways Altered in Response to Vibration in a Model of Hand-Arm Vibration Syndrome.

PubMed

Waugh, Stacey; Kashon, Michael L; Li, Shengqiao; Miller, Gerome R; Johnson, Claud; Krajnak, Kristine

2016-04-01

The aim of this study was to use an established model of vibration-induced injury to assess frequency-dependent changes in transcript expression in skin, artery, and nerve tissues. Transcript expression in tissues from control and vibration-exposed rats (4 h/day for 10 days at 62.5, 125, or 250 Hz; 49 m/s, rms) was measured. Transcripts affected by vibration were used in bioinformatics analyses to identify molecular- and disease-related pathways associated with exposure to vibration. Analyses revealed that cancer-related pathways showed frequency-dependent changes in activation or inhibition. Most notably, the breast-related cancer-1 pathway was affected. Other pathways associated with breast cancer type 1 susceptibility protein related signaling, or associated with cancer and cell cycle/cell survivability were also affected. Occupational exposure to vibration may result in DNA damage and alterations in cell signaling pathways that have significant effects on cellular division.

MEK-Dependent Negative Feedback Underlies BCR-ABL-Mediated Oncogene Addiction

PubMed Central

Asmussen, Jennifer; Lasater, Elisabeth A.; Tajon, Cheryl; Oses-Prieto, Juan; Jun, Young-wook; Taylor, Barry S.; Burlingame, Alma; Craik, Charles S.; Shah, Neil P.

2014-01-01

The clinical experience with BCR-ABL tyrosine kinase inhibitors (TKIs) for the treatment of chronic myeloid leukemia (CML) provides compelling evidence for oncogene addiction. Yet, the molecular basis of oncogene addiction remains elusive. Through unbiased quantitative phosphoproteomic analyses of CML cells transiently exposed to BCR-ABL TKI, we identified persistent downregulation of growth factor receptor (GF-R) signaling pathways. We then established and validated a tissue-relevant isogenic model of BCR-ABL-mediated addiction, and found evidence for myeloid GF-R signaling pathway rewiring that profoundly and persistently dampens physiologic pathway activation. We demonstrate that eventual restoration of ligand-mediated GF-R pathway activation is insufficient to fully rescue cells from a competing apoptotic fate. In contrast to previous work with BRAFV600E in melanoma cells, feedback inhibition following BCR-ABL TKI treatment is markedly prolonged, extending beyond the time required to initiate apoptosis. Mechanistically, BCR-ABL-mediated oncogene addiction is facilitated by persistent high levels of MEK-dependent negative feedback. PMID:24362263

Disturbed MEK/ERK signaling increases osteoclast activity via the Hedgehog-Gli pathway in postmenopausal osteoporosis.

PubMed

Li, Xiaojie; Jie, Qiang; Zhang, Hongyang; Zhao, Yantao; Lin, Yangjing; Du, Junjie; Shi, Jun; Wang, Long; Guo, Kai; Li, Yong; Wang, Chunhui; Gao, Bo; Huang, Qiang; Liu, Jian; Yang, Liu; Luo, Zhuojing

2016-11-01

Postmenopausal osteoporosis is a worldwide health problem and is characterized by increased and activated osteoclasts. However, the mechanism by which osteoclasts are dysregulated in postmenopausal osteoporosis is not fully understood. In this study, we found that the Hedgehog-Gli pathway was upregulated in postmenopausal osteoporotic osteoclasts and that 17β-estradiol both inhibited osteoclastogenesis and induced osteoclast apoptosis by downregulating Hedgehog-Gli signaling. Furthermore, we demonstrated that the Hedgehog-Gli pathway was negatively regulated by MEK/ERK signaling and that this effect was Sonic Hedgehog (SHH)-dependent and was partially blocked by an anti-SHH antibody. Moreover, we found that the stimulatory effect of Hedgehog signaling on osteoclastogenesis and the inhibitory effect on osteoclast apoptosis were dependent on the Gli family of transcription factors. The pathways and molecules that contribute to the regulation of osteoclastogenesis and apoptosis represent potential new strategies for designing molecular drugs for the treatment of postmenopausal osteoporosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Constitutively Active Akt Induces Ectodermal Defects and Impaired Bone Morphogenetic Protein Signaling

PubMed Central

Segrelles, Carmen; Moral, Marta; Lorz, Corina; Santos, Mirentxu; Lu, Jerry; Cascallana, José Luis; Lara, M. Fernanda; Carbajal, Steve; Martínez-Cruz, Ana Belén; García-Escudero, Ramón; Beltran, Linda; Segovia, José C.; Bravo, Ana

2008-01-01

Aberrant activation of the Akt pathway has been implicated in several human pathologies including cancer. However, current knowledge on the involvement of Akt signaling in development is limited. Previous data have suggested that Akt-mediated signaling may be an essential mediator of epidermal homeostasis through cell autonomous and noncell autonomous mechanisms. Here we report the developmental consequences of deregulated Akt activity in the basal layer of stratified epithelia, mediated by the expression of a constitutively active Akt1 (myrAkt) in transgenic mice. Contrary to mice overexpressing wild-type Akt1 (Aktwt), these myrAkt mice display, in a dose-dependent manner, altered development of ectodermally derived organs such as hair, teeth, nails, and epidermal glands. To identify the possible molecular mechanisms underlying these alterations, gene profiling approaches were used. We demonstrate that constitutive Akt activity disturbs the bone morphogenetic protein-dependent signaling pathway. In addition, these mice also display alterations in adult epidermal stem cells. Collectively, we show that epithelial tissue development and homeostasis is dependent on proper regulation of Akt expression and activity. PMID:17959825

Radiation and inhibition of angiogenesis by canstatin synergize to induce HIF-1α–mediated tumor apoptotic switch

PubMed Central

Magnon, Claire; Opolon, Paule; Ricard, Marcel; Connault, Elisabeth; Ardouin, Patrice; Galaup, Ariane; Métivier, Didier; Bidart, Jean-Michel; Germain, Stéphane; Perricaudet, Michel; Schlumberger, Martin

2007-01-01

Tumor radioresponsiveness depends on endothelial cell death, which leads in turn to tumor hypoxia. Radiation-induced hypoxia was recently shown to trigger tumor radioresistance by activating angiogenesis through hypoxia-inducible factor 1–regulated (HIF-1–regulated) cytokines. We show here that combining targeted radioiodide therapy with angiogenic inhibitors, such as canstatin, enhances direct tumor cell apoptosis, thereby overcoming radio-induced HIF-1–dependent tumor survival pathways in vitro and in vivo. We found that following dual therapy, HIF-1α increases the activity of the canstatin-induced αvβ5 signaling tumor apoptotic pathway and concomitantly abrogates mitotic checkpoint and tetraploidy triggered by radiation. Apoptosis in conjunction with mitotic catastrophe leads to lethal tumor damage. We discovered that HIF-1 displays a radiosensitizing activity that is highly dependent on treatment modalities by regulating key apoptotic molecular pathways. Our findings therefore support a crucial role for angiogenesis inhibitors in shifting the fate of radiation-induced HIF-1α activity from hypoxia-induced tumor radioresistance to hypoxia-induced tumor apoptosis. This study provides a basis for developing new biology-based clinically relevant strategies to improve the efficacy of radiation oncology, using HIF-1 as an ally for cancer therapy. PMID:17557121

Potassium efflux fires the canon: Potassium efflux as a common trigger for canonical and noncanonical NLRP3 pathways.

PubMed

Rivers-Auty, Jack; Brough, David

2015-10-01

Murine caspase-11 and its human orthologues, caspase-4 and caspase-5, activate an inflammatory response following cytoplasmic recognition of cell wall constituents from Gram-negative bacteria, such as LPS. This inflammatory response involves pyroptotic cell death and the concomitant release of IL-1α, as well as the production of IL-1β and IL-18 through the noncanonical NLR family, pyrin domain containing 3 (NLRP3) pathway. This commentary discusses three papers in this issue of the European Journal of Immunology that advance our understanding of the roles of caspase-11, -4, and -5 in the noncanonical pathway. By utilizing the new gene editing technique, clustered regularly interspaced short palindromic repeats (CRISPR), as well as sensitive cell imaging techniques, these papers establish that cytoplasmic LPS-dependent IL-1β production requires the NLRP3 inflammasome and that its activation is dependent on K(+) efflux, whereas IL-1α release and pyroptotic cell death pathways are NLRP3-independent. These findings expand on previous research implicating K(+) efflux as the principal trigger for NLRP3 activation and suggest that canonical and noncanonical NLRP3 pathways are not as dissimilar as first thought. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Alternative Epac/cAMP Pathway and the MAPK Pathway Mediate hCG Induction of Leptin in Placental Cells

PubMed Central

Maymó, Julieta Lorena; Pérez Pérez, Antonio; Maskin, Bernardo; Dueñas, José Luis; Calvo, Juan Carlos; Sánchez Margalet, Víctor; Varone, Cecilia Laura

2012-01-01

Pleiotropic effects of leptin have been identified in reproduction and pregnancy, particularly in the placenta, where it works as an autocrine hormone. In this work, we demonstrated that human chorionic gonadotropin (hCG) added to JEG-3 cell line or to placental explants induces endogenous leptin expression. We also found that hCG increased cAMP intracellular levels in BeWo cells in a dose-dependent manner, stimulated cAMP response element (CRE) activity and the cotransfection with an expression plasmid of a dominant negative mutant of CREB caused a significant inhibition of hCG stimulation of leptin promoter activity. These results demonstrate that hCG indeed activates cAMP/PKA pathway, and that this pathway is involved in leptin expression. Nevertheless, we found leptin induction by hCG is dependent on cAMP levels. Treatment with (Bu)2cAMP in combination with low and non stimulatory hCG concentrations led to an increase in leptin expression, whereas stimulatory concentrations showed the opposite effect. We found that specific PKA inhibition by H89 caused a significant increase of hCG leptin induction, suggesting that probably high cAMP levels might inhibit hCG effect. It was found that hCG enhancement of leptin mRNA expression involved the MAPK pathway. In this work, we demonstrated that hCG leptin induction through the MAPK signaling pathway is inhibited by PKA. We observed that ERK1/2 phosphorylation increased when hCG treatment was combined with H89. In view of these results, the involvement of the alternative cAMP/Epac signaling pathway was studied. We observed that a cAMP analogue that specifically activates Epac (CPT-OMe) stimulated leptin expression by hCG. In addition, the overexpression of Epac and Rap1 proteins increased leptin promoter activity and enhanced hCG. In conclusion, we provide evidence suggesting that hCG induction of leptin gene expression in placenta is mediated not only by activation of the MAPK signaling pathway but also by the alternative cAMP/Epac signaling pathway. PMID:23056265

Shiga Toxins Activate the NLRP3 Inflammasome Pathway To Promote Both Production of the Proinflammatory Cytokine Interleukin-1β and Apoptotic Cell Death

PubMed Central

Lee, Moo-Seung; Kwon, Haenaem; Lee, Eun-Young; Kim, Dong-Jae; Park, Jong-Hwan; Tesh, Vernon L.; Oh, Tae-Kwang

2015-01-01

Shiga toxin (Stx)-mediated immune responses, including the production of the proinflammatory cytokines tumor necrosis-α (TNF-α) and interleukin-1β (IL-1β), may exacerbate vascular damage and accelerate lethality. However, the immune signaling pathway activated in response to Stx is not well understood. Here, we demonstrate that enzymatically active Stx, which leads to ribotoxic stress, triggers NLRP3 inflammasome-dependent caspase-1 activation and IL-1β secretion in differentiated macrophage-like THP-1 (D-THP-1) cells. The treatment of cells with a chemical inhibitor of glycosphingolipid biosynthesis, which suppresses the expression of the Stx receptor globotriaosylceramide and subsequent endocytosis of the toxin, substantially blocked activation of the NLRP3 inflammasome and processing of caspase-1 and IL-1β. Processing and release of both caspase-1 and IL-1β were significantly reduced or abolished in Stx-intoxicated D-THP-1 cells in which the expression of NLRP3 or ASC was stably knocked down. Furthermore, Stx mediated the activation of caspases involved in apoptosis in an NLRP3- or ASC-dependent manner. In Stx-intoxicated cells, the NLRP3 inflammasome triggered the activation of caspase-8/3, leading to the initiation of apoptosis, in addition to caspase-1-dependent pyroptotic cell death. Taken together, these results suggest that Stxs trigger the NLRP3 inflammasome pathway to release proinflammatory IL-1β as well as to promote apoptotic cell death. PMID:26502906

Adiponectin promotes hyaluronan synthesis along with increases in hyaluronan synthase 2 transcripts through an AMP-activated protein kinase/peroxisome proliferator-activated receptor-{alpha}-dependent pathway in human dermal fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamane, Takumi; Kobayashi-Hattori, Kazuo; Oishi, Yuichi, E-mail: y3oishi@nodai.ac.jp

2011-11-18

Highlights: Black-Right-Pointing-Pointer Adiponectin promotes hyaluronan synthesis along with an increase in HAS2 transcripts. Black-Right-Pointing-Pointer Adiponectin also increases the phosphorylation of AMPK. Black-Right-Pointing-Pointer A pharmacological activator of AMPK increases mRNA levels of PPAR{alpha} and HAS2. Black-Right-Pointing-Pointer Adiponectin-induced HAS2 mRNA expression is blocked by a PPAR{alpha} antagonist. Black-Right-Pointing-Pointer Adiponectin promotes hyaluronan synthesis via an AMPK/PPAR{alpha}-dependent pathway. -- Abstract: Although adipocytokines affect the functions of skin, little information is available on the effect of adiponectin on the skin. In this study, we investigated the effect of adiponectin on hyaluronan synthesis and its regulatory mechanisms in human dermal fibroblasts. Adiponectin promoted hyaluronan synthesis alongmore » with an increase in the mRNA levels of hyaluronan synthase 2 (HAS2), which plays a primary role in hyaluronan synthesis. Adiponectin also increased the phosphorylation of AMP-activated protein kinase (AMPK). A pharmacological activator of AMPK, 5-aminoimidazole-4-carboxamide-1{beta}-ribofuranoside (AICAR), increased mRNA levels of peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}), which enhances the expression of HAS2 mRNA. In addition, AICAR increased the mRNA levels of HAS2. Adiponectin-induced HAS2 mRNA expression was blocked by GW6471, a PPAR{alpha} antagonist, in a concentration-dependent manner. These results show that adiponectin promotes hyaluronan synthesis along with increases in HAS2 transcripts through an AMPK/PPAR{alpha}-dependent pathway in human dermal fibroblasts. Thus, our study suggests that adiponectin may be beneficial for retaining moisture in the skin, anti-inflammatory activity, and the treatment of a variety of cutaneous diseases.« less

Curcuminoid Binding to Embryonal Carcinoma Cells: Reductive Metabolism, Induction of Apoptosis, Senescence, and Inhibition of Cell Proliferation

PubMed Central

Quitschke, Wolfgang W.

2012-01-01

Curcumin preparations typically contain a mixture of polyphenols, collectively referred to as curcuminoids. In addition to the primary component curcumin, they also contain smaller amounts of the co-extracted derivatives demethoxycurcumin and bisdemethoxycurcumin. Curcuminoids can be differentially solubilized in serum, which allows for the systematic analysis of concentration-dependent cellular binding, biological effects, and metabolism. Technical grade curcumin was solubilized in fetal calf serum by two alternative methods yielding saturated preparations containing either predominantly curcumin (60%) or bisdemethoxycurcumin (55%). Continual exposure of NT2/D1 cells for 4–6 days to either preparation in cell culture media reduced cell division (1–5 µM), induced senescence (6–7 µM) or comprehensive cell death (8–10 µM) in a concentration-dependent manner. Some of these effects could also be elicited in cells transiently exposed to higher concentrations of curcuminoids (47 µM) for 0.5–4 h. Curcuminoids induced apoptosis by generalized activation of caspases but without nucleosomal fragmentation. The equilibrium binding of serum-solubilized curcuminoids to NT2/D1 cells incubated with increasing amounts of curcuminoid-saturated serum occurred with apparent overall dissociation constants in the 6–10 µM range. However, the presence of excess free serum decreased cellular binding in a hyperbolic manner. Cellular binding was overwhelmingly associated with membrane fractions and bound curcuminoids were metabolized in NT2/D1 cells via a previously unidentified reduction pathway. Both the binding affinities for curcuminoids and their reductive metabolic pathways varied in other cell lines. These results suggest that curcuminoids interact with cellular binding sites, thereby activating signal transduction pathways that initiate a variety of biological responses. The dose-dependent effects of these responses further imply that distinct cellular pathways are sequentially activated and that this activation is dependent on the affinity of curcuminoids for the respective binding sites. Defined serum-solubilized curcuminoids used in cell culture media are thus suitable for further investigating the differential activation of signal transduction pathways. PMID:22768090

A Myc-driven self-reinforcing regulatory network maintains mouse embryonic stem cell identity

PubMed Central

Fagnocchi, Luca; Cherubini, Alessandro; Hatsuda, Hiroshi; Fasciani, Alessandra; Mazzoleni, Stefania; Poli, Vittoria; Berno, Valeria; Rossi, Riccardo L.; Reinbold, Rolland; Endele, Max; Schroeder, Timm; Rocchigiani, Marina; Szkarłat, Żaneta; Oliviero, Salvatore; Dalton, Stephen; Zippo, Alessio

2016-01-01

Stem cell identity depends on the integration of extrinsic and intrinsic signals, which directly influence the maintenance of their epigenetic state. Although Myc transcription factors play a major role in stem cell self-renewal and pluripotency, their integration with signalling pathways and epigenetic regulators remains poorly defined. We addressed this point by profiling the gene expression and epigenetic pattern in ESCs whose growth depends on conditional Myc activity. Here we show that Myc potentiates the Wnt/β-catenin signalling pathway, which cooperates with the transcriptional regulatory network in sustaining ESC self-renewal. Myc activation results in the transcriptional repression of Wnt antagonists through the direct recruitment of PRC2 on these targets. The consequent potentiation of the autocrine Wnt/β-catenin signalling induces the transcriptional activation of the endogenous Myc family members, which in turn activates a Myc-driven self-reinforcing circuit. Thus, our data unravel a Myc-dependent self-propagating epigenetic memory in the maintenance of ESC self-renewal capacity. PMID:27301576

A Myc-driven self-reinforcing regulatory network maintains mouse embryonic stem cell identity.

PubMed

Fagnocchi, Luca; Cherubini, Alessandro; Hatsuda, Hiroshi; Fasciani, Alessandra; Mazzoleni, Stefania; Poli, Vittoria; Berno, Valeria; Rossi, Riccardo L; Reinbold, Rolland; Endele, Max; Schroeder, Timm; Rocchigiani, Marina; Szkarłat, Żaneta; Oliviero, Salvatore; Dalton, Stephen; Zippo, Alessio

2016-06-15

Stem cell identity depends on the integration of extrinsic and intrinsic signals, which directly influence the maintenance of their epigenetic state. Although Myc transcription factors play a major role in stem cell self-renewal and pluripotency, their integration with signalling pathways and epigenetic regulators remains poorly defined. We addressed this point by profiling the gene expression and epigenetic pattern in ESCs whose growth depends on conditional Myc activity. Here we show that Myc potentiates the Wnt/β-catenin signalling pathway, which cooperates with the transcriptional regulatory network in sustaining ESC self-renewal. Myc activation results in the transcriptional repression of Wnt antagonists through the direct recruitment of PRC2 on these targets. The consequent potentiation of the autocrine Wnt/β-catenin signalling induces the transcriptional activation of the endogenous Myc family members, which in turn activates a Myc-driven self-reinforcing circuit. Thus, our data unravel a Myc-dependent self-propagating epigenetic memory in the maintenance of ESC self-renewal capacity.

Structures of apicomplexan calcium-dependent protein kinases reveal mechanism of activation by calcium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wernimont, Amy K; Artz, Jennifer D.; Jr, Patrick Finerty

2010-09-21

Calcium-dependent protein kinases (CDPKs) have pivotal roles in the calcium-signaling pathway in plants, ciliates and apicomplexan parasites and comprise a calmodulin-dependent kinase (CaMK)-like kinase domain regulated by a calcium-binding domain in the C terminus. To understand this intramolecular mechanism of activation, we solved the structures of the autoinhibited (apo) and activated (calcium-bound) conformations of CDPKs from the apicomplexan parasites Toxoplasma gondii and Cryptosporidium parvum. In the apo form, the C-terminal CDPK activation domain (CAD) resembles a calmodulin protein with an unexpected long helix in the N terminus that inhibits the kinase domain in the same manner as CaMKII. Calcium bindingmore » triggers the reorganization of the CAD into a highly intricate fold, leading to its relocation around the base of the kinase domain to a site remote from the substrate binding site. This large conformational change constitutes a distinct mechanism in calcium signal-transduction pathways.« less

Cannabinoid transmission in the prelimbic cortex bidirectionally controls opiate reward and aversion signaling through dissociable kappa versus μ-opiate receptor dependent mechanisms.

PubMed

Ahmad, Tasha; Lauzon, Nicole M; de Jaeger, Xavier; Laviolette, Steven R

2013-09-25

Cannabinoid, dopamine (DA), and opiate receptor pathways play integrative roles in emotional learning, associative memory, and sensory perception. Modulation of cannabinoid CB1 receptor transmission within the medial prefrontal cortex (mPFC) regulates the emotional valence of both rewarding and aversive experiences. Furthermore, CB1 receptor substrates functionally interact with opiate-related motivational processing circuits, particularly in the context of reward-related learning and memory. Considerable evidence demonstrates functional interactions between CB1 and DA signaling pathways during the processing of motivationally salient information. However, the role of mPFC CB1 receptor transmission in the modulation of behavioral opiate-reward processing is not currently known. Using an unbiased conditioned place preference paradigm with rats, we examined the role of intra-mPFC CB1 transmission during opiate reward learning. We report that activation or inhibition of CB1 transmission within the prelimbic cortical (PLC) division of the mPFC bidirectionally regulates the motivational valence of opiates; whereas CB1 activation switched morphine reward signaling into an aversive stimulus, blockade of CB1 transmission potentiated the rewarding properties of normally sub-reward threshold conditioning doses of morphine. Both of these effects were dependent upon DA transmission as systemic blockade of DAergic transmission prevented CB1-dependent modulation of morphine reward and aversion behaviors. We further report that CB1-mediated intra-PLC opiate motivational signaling is mediated through a μ-opiate receptor-dependent reward pathway, or a κ-opiate receptor-dependent aversion pathway, directly within the ventral tegmental area. Our results provide evidence for a novel CB1-mediated motivational valence switching mechanism within the PLC, controlling dissociable subcortical reward and aversion pathways.

Sirt1 negatively regulates FcεRI-mediated mast cell activation through AMPK- and PTP1B-dependent processes.

PubMed

Li, Xian; Lee, Youn Ju; Jin, Fansi; Park, Young Na; Deng, Yifeng; Kang, Youra; Yang, Ju Hye; Chang, Jae-Hoon; Kim, Dong-Young; Kim, Jung-Ae; Chang, Young-Chae; Ko, Hyun-Jeong; Kim, Cheorl-Ho; Murakami, Makoto; Chang, Hyeun Wook

2017-07-25

Sirt1, a key regulator of metabolism and longevity, has recently been implicated in the regulation of allergic reactions, although the underlying mechanism remains unclear. Here we show that Sirt1 negatively regulates FcεRI-stimulated mast cell activation and anaphylaxis through two mutually regulated pathways involving AMP-activated protein kinase (AMPK) and protein tyrosine phosphatase 1B (PTP1B). Mast cell-specific knockout of Sirt1 dampened AMPK-dependent suppression of FcεRI signaling, thereby augmenting mast cell activation both in vitro and in vivo. Sirt1 inhibition of FcεRI signaling also involved an alternative component, PTP1B, which attenuated the inhibitory AMPK pathway and conversely enhanced the stimulatory Syk pathway, uncovering a novel role of this phosphatase. Moreover, a Sirt1 activator resveratrol stimulated the inhibitory AMPK axis, with reciprocal suppression of the stimulatory PTP1B/Syk axis, thus potently inhibiting anaphylaxis. Overall, our results provide a molecular explanation for the beneficial role of Sirt1 in allergy and underscore a potential application of Sirt1 activators as a new class of anti-allergic agents.

PI3K is required for both basal and LPA-induced DNA synthesis in oral carcinoma cells.

PubMed

Aasrum, Monica; Tjomsland, Vegard; Thoresen, G Hege; De Angelis, Paula M; Christoffersen, Thoralf; Brusevold, Ingvild J

2016-07-01

The glycerophospholipid lysophosphatidic acid (LPA), which is present in most tissues and in high concentrations in saliva, may exert profound effects on oral cancer cells. We have investigated mitogenic signalling induced by LPA in the two oral carcinoma cell lines, D2 and E10, focusing on the role of EGFR transactivation and downstream pathways. Two oral squamous carcinoma cell lines, D2 and E10, were analysed for effects of LPA on signalling pathways and induction of DNA synthesis. Pathway activation was investigated by examining phosphorylation of signalling proteins and by the use of specific pathway inhibitors. The D2 cells had higher levels of activated signalling proteins and higher DNA synthesis activity in the basal condition than E10 cells. EGF did not induce proliferation in D2 cells, whereas LPA induced proliferation in both cell lines, by mechanisms depending on EGFR transactivation. Release of EGFR ligands was involved in basal and LPA-induced proliferation in both D2 and E10 cells. The proliferation in D2 cells was dependent on the PI3K/Akt pathway, but not the MEK/ERK pathway. In E10 cells, the PI3K/Akt, MEK/ERK and p38 pathways were all involved in the proliferation. Transactivation of EGFR is required for LPA-induced DNA synthesis in D2 and E10 cells. Our results also show that although proliferation of oral carcinoma cells is regulated by several pathways, and differentially in E10 and D2 cells, the PI3K pathway has a crucial role in both cell lines. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Effects of Neonicotinoid Pesticides on Promoter-Specific Aromatase (CYP19) Expression in Hs578t Breast Cancer Cells and the Role of the VEGF Pathway.

PubMed

Caron-Beaudoin, Élyse; Viau, Rachel; Sanderson, J Thomas

2018-04-26

Aromatase (CYP19) is a key enzyme in estrogens biosynthesis. In the mammary gland, CYP19 gene is expressed at low levels under the regulation of its I.4 promoter. In hormone-dependent breast cancer, fibroblast cells surrounding the tumor express increased levels of CYP19 mRNA due to a decrease of I.4 promoter activity and an increase of PII, I.3, and I.7 promoter activity. Little is known about the effects of environmental chemicals on the promoter-specific CYP19 expression. We aimed to determine the effects of two neonicotinoids (thiacloprid and imidacloprid) on promoter-specific CYP19 expression in Hs578t breast cancer cells and understand the signaling pathways involved. Hs578t cells were exposed to various signaling pathway stimulants or neonicotinoids for 24 h. Promoter-specific expression of CYP19 was determined by real-time quantitative polymerase chain reaction and catalytic activity of aromatase by tritiated water release assay. To our knowledge, we are the first to demonstrate that the normal I.4 promoter and the breast cancer-relevant PII, I.3, and I.7 promoters of CYP19 are active in these cells. We found that the expression of CYP19 via promoters PII, I.3, and I.7 in Hs578t cells was, in part, dependent on the activation of two VEGF signaling pathways: mitogen-activated protein kinase (MAPK) 1/3 and phospholipase C (PLC). Exposure of Hs578t cells to environmental concentrations of imidacloprid and thiacloprid resulted in a switch in CYP19 promoter usage, involving inhibition of I.4 promoter activity and an increase of PII, I.3, and I.7 promoter-mediated CYP19 expression and aromatase catalytic activity. Greater effects were seen at lower concentrations. Our results suggest that thiacloprid and imidacloprid exert their effects at least partially by inducing the MAPK 1/3 and/or PLC pathways. We demonstrated in vitro that neonicotinoids may stimulate a change in CYP19 promoter usage similar to that observed in patients with hormone-dependent breast cancer. https://doi.org/10.1289/EHP2698.

2',5'-Dihydroxychalcone-induced glutathione is mediated by oxidative stress and kinase signaling pathways.

PubMed

Kachadourian, Remy; Pugazhenthi, Subbiah; Velmurugan, Kalpana; Backos, Donald S; Franklin, Christopher C; McCord, Joe M; Day, Brian J

2011-09-15

Hydroxychalcones are naturally occurring compounds that continue to attract considerable interest because of their anti-inflammatory and antiangiogenic properties. They have been reported to inhibit the synthesis of the inducible nitric oxide synthase and to induce the expression of heme oxygenase-1. This study examines the mechanisms by which 2',5'-dihydroxychalcone (2',5'-DHC) induces an increase in cellular glutathione (GSH) levels using a cell line stably expressing a luciferase reporter gene driven by antioxidant-response elements (MCF-7/AREc32). The 2',5'-DHC-induced increase in cellular GSH levels was partially inhibited by the catalytic antioxidant MnTDE-1,3-IP(5+), suggesting that reactive oxygen species (ROS) mediate the antioxidant adaptive response. 2',5'-DHC treatment induced phosphorylation of the c-Jun N-terminal kinase (JNK) pathway, which was also inhibited by MnTDE-1,3-IP(5+). These findings suggest a ROS-dependent activation of the AP-1 transcriptional response. However, whereas 2',5'-DHC triggered the NF-E2-related factor 2 (Nrf2) transcriptional response, cotreatment with MnTDE-1,3-IP(5+) did not decrease 2',5'-DHC-induced Nrf2/ARE activity, showing that this pathway is not dependent on ROS. Moreover, pharmacological inhibitors of mitogen-activated protein kinase (MAPK) pathways showed a role for JNK and p38MAPK in mediating the 2',5'-DHC-induced Nrf2 response. These findings suggest that the 2',5'-DHC-induced increase in GSH levels results from a combination of ROS-dependent and ROS-independent pathways. Copyright © 2011 Elsevier Inc. All rights reserved.

Saccharomyces cerevisiae Ras/cAMP pathway controls post-diauxic shift element-dependent transcription through the zinc finger protein Gis1

PubMed Central

Pedruzzi, Ivo; Bürckert, Niels; Egger, Pascal; De Virgilio, Claudio

2000-01-01

The Saccharomyces cerevisiae protein kinase Rim15 was identified previously as a component of the Ras/cAMP pathway acting immediately downstream of cAMP-dependent protein kinase (cAPK) to control a broad range of adaptations in response to nutrient limitation. Here, we show that the zinc finger protein Gis1 acts as a dosage-dependent suppressor of the rim15Δ defect in nutrient limitation-induced transcriptional derepression of SSA3. Loss of Gis1 results in a defect in transcriptional derepression upon nutrient limitation of various genes that are negatively regulated by the Ras/cAMP pathway (e.g. SSA3, HSP12 and HSP26). Tests of epistasis as well as transcriptional analyses of Gis1-dependent expression indicate that Gis1 acts in this pathway downstream of Rim15 to mediate transcription from the previously identified post-diauxic shift (PDS) element. Accordingly, deletion of GIS1 partially suppresses, and overexpression of GIS1 exacerbates the growth defect of mutant cells that are compromised for cAPK activity. Moreover, PDS element-driven expression, which is negatively regulated by the Ras/cAMP pathway and which is induced upon nutrient limitation, is almost entirely dependent on the presence of Gis1. PMID:10835355

AMPK activators inhibit the proliferation of human melanomas bearing the activated MAPK pathway.

PubMed

Petti, Carlotta; Vegetti, Claudia; Molla, Alessandra; Bersani, Ilaria; Cleris, Loredana; Mustard, Kirsty J; Formelli, Franca; Hardie, Grahame D; Sensi, Marialuisa; Anichini, Andrea

2012-10-01

Raf/MEK/ERK signaling can inhibit the liver kinase B1-AMP-activated protein kinase (LKB1-AMPK) pathway, thus rendering melanoma cells resistant to energy stress conditions. We evaluated whether pharmacological reactivation of the AMPK function could exert antitumor effects on melanoma cells bearing this pathway constitutively active because of a mutation in NRAS or BRAF genes. Nine melanoma cell lines were treated with the AMPK activators 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR) and phenformin. The activation of AMPK enzymatic activity, phosphorylation of AMPK and acetyl-CoA carboxylase kinase, in-vitro proliferation, cell cycle, and in-vivo growth of xenografts in nude mice were evaluated. AICAR and phenformin promoted phosphorylation and enzymatic activity of AMPK, as well as phosphorylation of the AMPK downstream target acetyl-CoA carboxylase. Drug treatment of either BRAF-mutant or NRAS-mutant melanomas, at doses not inducing cell death, was accompanied by a dose-dependent decrease in melanoma cell proliferation because of cell cycle arrest in either the G0/G1 or the S phase, associated with an increased expression of the p21 cell cycle inhibitor. Melanomas isolated from subcutaneously implanted mice, 25 days from treatment with AICAR, showed increased staining of the senescence-associated marker β-galactosidase, high p21 expression, and evidence of necrosis. Altogether, these results indicate that pharmacological activators of AMPK-dependent pathways inhibit the cell growth of melanoma cells with active Raf/MEK/ERK signaling and provide a rationale for further investigation on their use in combination therapies.

Involvement of PI3K/AKT and MAPK Pathways for TNF-α Production in SiHa Cervical Mucosal Epithelial Cells Infected with Trichomonas vaginalis.

PubMed

Yang, Jung-Bo; Quan, Juan-Hua; Kim, Ye-Eun; Rhee, Yun-Ee; Kang, Byung-Hyun; Choi, In-Wook; Cha, Guang-Ho; Yuk, Jae-Min; Lee, Young-Ha

2015-08-01

Trichomonas vaginalis; induces proinflammation in cervicovaginal mucosal epithelium. To investigate the signaling pathways in TNF-α production in cervical mucosal epithelium after T. vaginalis infection, the phosphorylation of PI3K/AKT and MAPK pathways were evaluated in T. vaginalis-infected SiHa cells in the presence and absence of specific inhibitors. T. vaginalis increased TNF-α production in SiHa cells, in a parasite burden-dependent and incubation time-dependent manner. In T. vaginalis-infected SiHa cells, AKT, ERK1/2, p38 MAPK, and JNK were phosphorylated from 1 hr after infection; however, the phosphorylation patterns were different from each other. After pretreatment with inhibitors of the PI3K/AKT and MAPK pathways, TNF-α production was significantly decreased compared to the control; however, TNF-α reduction patterns were different depending on the type of PI3K/MAPK inhibitors. TNF-α production was reduced in a dose-dependent manner by treatment with wortmannin and PD98059, whereas it was increased by SP600125. These data suggested that PI3K/AKT and MAPK signaling pathways are important in regulation of TNF-α production in cervical mucosal epithelial SiHa cells. However, activation patterns of each pathway were different from the types of PI3K/MAPK pathways.

GATA-dependent regulation of TPO-induced c-mpl gene expression during megakaryopoiesis.

PubMed

Sunohara, Masataka; Morikawa, Shigeru; Fuse, Akira; Sato, Iwao

2014-01-01

Thrombopoietin (TPO) and its receptor, c-Mpl, play the crucial role during megakaryocytopoiesis. Previously, we have shown that the promoter activity of c-mpl induced by TPO is modulated by transcription through a PKC-dependent pathway and that GATA(-77) is involved as a positive regulatory element in TPO-induced c-mpl gene expression in the megakaryoblastic CMK cells. In this research, to examine participating possibility of GATA promoter element in TPO- induced c-mpl gene expression through a PKC-independent pathway, the promoter activity of site-directed mutagenesis and the effect of potein kinase C modulator were measured by a transient transfection assay system. Together with our previous results on the TPO-induced c-mpl promoter, this study indicates destruction of -77GATA in c-mpl promoter decreased the activity by 47.3% under existence of GF109203. These results suggest that GATA promoter element plays significant role in TPO-induced c-mpl gene expression through a PKC-independent pathway.

Astrocytes mediate synapse elimination through MEGF10 and MERTK pathways

NASA Astrophysics Data System (ADS)

Chung, Won-Suk; Clarke, Laura E.; Wang, Gordon X.; Stafford, Benjamin K.; Sher, Alexander; Chakraborty, Chandrani; Joung, Julia; Foo, Lynette C.; Thompson, Andrew; Chen, Chinfei; Smith, Stephen J.; Barres, Ben A.

2013-12-01

To achieve its precise neural connectivity, the developing mammalian nervous system undergoes extensive activity-dependent synapse remodelling. Recently, microglial cells have been shown to be responsible for a portion of synaptic pruning, but the remaining mechanisms remain unknown. Here we report a new role for astrocytes in actively engulfing central nervous system synapses. This process helps to mediate synapse elimination, requires the MEGF10 and MERTK phagocytic pathways, and is strongly dependent on neuronal activity. Developing mice deficient in both astrocyte pathways fail to refine their retinogeniculate connections normally and retain excess functional synapses. Finally, we show that in the adult mouse brain, astrocytes continuously engulf both excitatory and inhibitory synapses. These studies reveal a novel role for astrocytes in mediating synapse elimination in the developing and adult brain, identify MEGF10 and MERTK as critical proteins in the synapse remodelling underlying neural circuit refinement, and have important implications for understanding learning and memory as well as neurological disease processes.

Differences in expression of genes in the MyD88 and TRIF signalling pathways and methylation of TLR4 and TRIF in Tibetan chickens and DaHeng S03 chickens infected with Salmonella enterica serovar enteritidis.

PubMed

Li, Xiaocheng; Zhang, Peng; Jiang, Xiaosong; Du, Huarui; Yang, Chaowu; Zhang, Zengrong; Men, Shuai; Zhang, Zhikun; Jiang, Wei; Wang, Hongning

2017-07-01

Salmonella enterica serovar (S. enteritidis) is a pathogenic bacterium that can cause symptoms of food poisoning, leading to death of poultry, resulting in serious economic losses. The MyD88 and TRIF signalling pathways play important roles in activating innate and adaptive immunity in chickens infected with S. enteritidis. The objective of the present study was to characterize in vivo mRNA expressions, protein levels and methylation levels of genes in the above two pathways in both Tibetan chickens and DaHeng S03 chickens infected with S. enteritidis. MyD88-dependent and TRIF-dependent signalling pathway were activated by infection, and the MyD88 signalling pathway induced cytokines LITAF and IL-8 played important roles in fighting against the S. enteritidis infection in vivo. The TLR4 methylation might alter expression of genes involved in the MyD88 signalling pathway, and thus different breeds of chickens might show differences in susceptibility to the S. enteritidis. The increased expression of INF β was activated by S. enteritidis, but its expressions were different in levels of mRNA and protein in Tibetan chickens and DaHeng chickens, suggesting its functions on the resistance to S. enteritidis infection in chickens. This study contributes to the understanding of two pathways activated in response to S. enteritidis infection, and gives indications on the mechanisms underlying resistance of Tibetan chickens and DaHeng chickens to S. enteritidis. Copyright © 2017 Elsevier B.V. All rights reserved.

w09, a novel autophagy enhancer, induces autophagy-dependent cell apoptosis via activation of the EGFR-mediated RAS-RAF1-MAP2K-MAPK1/3 pathway.

PubMed

Zhang, Pinghu; Zheng, Zuguo; Ling, Li; Yang, Xiaohui; Zhang, Ni; Wang, Xue; Hu, Maozhi; Xia, Yu; Ma, Yiwen; Yang, Haoran; Wang, Yunyi; Liu, Hongqi

2017-07-03

The EGFR (epidermal growth factor receptor) signaling pathway is frequently deregulated in many malignancies. Therefore, targeting the EGFR pathway is regarded as a promising strategy for anticancer drug discovery. Herein, we identified a 2-amino-nicotinonitrile compound (w09) as a novel autophagy enhancer, which potently induced macroautophagy/autophagy and consequent apoptosis in gastric cancer cells. Mechanistic studies revealed that EGFR-mediated activation of the RAS-RAF1-MAP2K-MAPK1/3 signaling pathway played a critical role in w09-induced autophagy and apoptosis of gastric cancer cells. Inhibition of the MAPK1/3 pathway with U0126 or blockade of autophagy by specific chemical inhibitors markedly attenuated the effect of w09-mediated growth inhibition and caspase-dependent apoptosis. Furthermore, these conclusions were supported by knockdown of ATG5 or knockout of ATG5 and/or ATG7. Notably, w09 increased the expression of SQSTM1 by transcription, and knockout of SQSTM1 or deleting the LC3-interaction region domain of SQSTM1, significantly inhibited w09-induced PARP1 cleavage, suggesting the central role played by SQSTM1 in w09-induced apoptosis. In addition, in vivo administration of w09 effectively inhibited tumor growth of SGC-7901 xenografts. Hence, our findings not only suggested that activation of the EGFR-RAS-RAF1-MAP2K-MAPK1/3 signaling pathway may play a critical role in w09-induced autophagy and apoptosis, but also imply that induction of autophagic cancer cell death through activation of the EGFR pathway may be a potential therapeutic strategy for EGFR-disregulated gastric tumors.

Molecular Targeting of Prostate Cancer During Androgen Ablation: Inhibition of CHES1/FOXN3

DTIC Science & Technology

2010-05-10

target of rapamycin ( mTOR ) and hypoxia-inducible factor-1α (HIF-1α) target genes (12). Additionally, transcriptional activation of Bcl-2 by NF-κB...in resistance to hormone therapy (19). Elevated expression of genes encoding PI3K/Akt/ mTOR pathway components has also been implicated in androgen... mTOR inhibition reverses Akt-dependent prostate intraepithelial neoplasia through regulation of apoptotic and HIF-1-dependent pathways. Nat Med, 10

Natural products induce a G protein-mediated calcium pathway activating p53 in cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ginkel, Paul R. van; Yan, Michael B.; Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, WI 53792

Paclitaxel, etoposide, vincristine and doxorubicin are examples of natural products being used as chemotherapeutics but with adverse side effects that limit their therapeutic window. Natural products derived from plants and having low toxicity, such as quercetin, resveratrol, epigallocatechin gallate and piceatannol, have been shown to inhibit tumor cell growth both in vitro and in pre-clinical models of cancer, but their mechanisms of action have not been fully elucidated, thus restricting their use as prototypes for developing synthetic analogs with improved anti-cancer properties. We and others have demonstrated that one of the earliest and consistent events upon exposure of tumor cellsmore » to these less toxic natural products is a rise in cytoplasmic calcium, activating several pro-apoptotic pathways. We describe here a G protein/inositol 1,4,5-trisphosphate pathway (InsP3) in MDA-MB-231 human breast cancer cells that mediates between these less toxic natural products and the release of calcium from the endoplasmic reticulum. Further, we demonstrate that this elevation of intracellular calcium modulates p53 activity and the subsequent transcription of several pro-apoptotic genes encoding PIG8, CD95, PIDD, TP53INP, RRM2B, Noxa, p21 and PUMA. We conclude from our findings that less toxic natural products likely bind to a G protein coupled receptor that activates a G protein-mediated and calcium-dependent pathway resulting selectively in tumor cell death. - Highlights: • Natural products having low toxicity increase cytoplasmic calcium in cancer cells. • A G-protein/IP{sub 3} pathway mediates the release of calcium from the ER. • The elevation of intracellular calcium modulates p53 activity. • p53 and other Ca{sup 2+}-dependent pro-apoptotic pathways inhibit cancer cell growth.« less

Factor X/Xa elicits protective signaling responses in endothelial cells directly via PAR-2 and indirectly via endothelial protein C receptor-dependent recruitment of PAR-1.

PubMed

Bae, Jong-Sup; Yang, Likui; Rezaie, Alireza R

2010-11-05

We recently demonstrated that the Gla domain-dependent interaction of protein C with endothelial protein C receptor (EPCR) leads to dissociation of the receptor from caveolin-1 and recruitment of PAR-1 to a protective signaling pathway. Thus, the activation of PAR-1 by either thrombin or PAR-1 agonist peptide elicited a barrier-protective response if endothelial cells were preincubated with protein C. In this study, we examined whether other vitamin K-dependent coagulation protease zymogens can modulate PAR-dependent signaling responses in endothelial cells. We discovered that the activation of both PAR-1 and PAR-2 in endothelial cells pretreated with factor FX (FX)-S195A, but not other procoagulant protease zymogens, also results in initiation of protective intracellular responses. Interestingly, similar to protein C, FX interaction with endothelial cells leads to dissociation of EPCR from caveolin-1 and recruitment of PAR-1 to a protective pathway. Further studies revealed that, FX activated by factor VIIa on tissue factor bearing endothelial cells also initiates protective signaling responses through the activation of PAR-2 independent of EPCR mobilization. All results could be recapitulated by the receptor agonist peptides to both PAR-1 and PAR-2. These results suggest that a cross-talk between EPCR and an unknown FX/FXa receptor, which does not require interaction with the Gla domain of FX, recruits PAR-1 to protective signaling pathways in endothelial cells.

Phenethyl isothiocyanate triggers apoptosis in human malignant melanoma A375.S2 cells through reactive oxygen species and the mitochondria-dependent pathways.

PubMed

Huang, S-H; Hsu, M-H; Hsu, S-C; Yang, J-S; Huang, W-W; Huang, A-C; Hsiao, Y-P; Yu, C-C; Chung, J-G

2014-03-01

We have reported previously that phenethyl isothiocyanate (PEITC) induces apoptosis in human osteosarcoma U-2 OS cells. Cytotoxic activity of PEITC towards other cancer cells such as human malignant melanoma and skin cancer cells has not been reported. In this study, the anticancer activity of PEITC towards human malignant melanoma cancer A375.S2 cells was investigated. To determine the mechanisms of PEITC inhibition of cell growth, the following end points were determined in A375.S2 cells: cell morphological changes, cell cycle arrest, DNA damage and fragmentation assays and morphological assessment of nuclear change, reactive oxygen species (ROS) and Ca(2+) generations, mitochondrial membrane potential disruption, and nitric oxide and 10-N-nonyl acridine orange productions, expression and activation of caspase-3 and -9, B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax), Bcl-2, poly (adenosine diphosphate-ribose) polymerase, and cytochrome c release, apoptosis-inducing factor and endonuclease G. PEITC induced morphological changes in time- and dose-dependent manner. PEITC induced G2/M phase arrest and induced apoptosis via endoplasmic reticulum stress-mediated mitochondria-dependent pathway. Western blot analysis showed that PEITC promoted Bax expression and inhibited Bcl-2 expression associated with the disintegration of the outer mitochondrial membrane causing cytochrome c release, and activation of caspase-9 and -3 cascade leading to apoptosis. We conclude that PEITC-triggered apoptotic death in A375.S2 cells occurs through ROS-mediated mitochondria-dependent pathways.

β-Hydroxy-β-Methylbutyrate (HMB) Promotes Neurite Outgrowth in Neuro2a Cells.

PubMed

Salto, Rafael; Vílchez, Jose D; Girón, María D; Cabrera, Elena; Campos, Nefertiti; Manzano, Manuel; Rueda, Ricardo; López-Pedrosa, Jose M

2015-01-01

β-Hydroxy-β-methylbutyrate (HMB) has been shown to enhance cell survival, differentiation and protein turnover in muscle, mainly activating phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) and mitogen-activated protein kinases/ extracellular-signal-regulated kinases (MAPK/ERK) signaling pathways. Since these two pathways are related to neuronal survival and differentiation, in this study, we have investigated the neurotrophic effects of HMB in mouse neuroblastoma Neuro2a cells. In Neuro2a cells, HMB promotes differentiation to neurites independent from any effects on proliferation. These effects are mediated by activation of both the PI3K/Akt and the extracellular-signal-regulated kinases (ERK1/2) signaling as demonstrated by the use of specific inhibitors of these two pathways. As myocyte-enhancer factor 2 (MEF2) family of transcription factors are involved in neuronal survival and plasticity, the transcriptional activity and protein levels of MEF2 were also evaluated. HMB promoted MEF2-dependent transcriptional activity mediated by the activation of Akt and ERK1/2 pathways. Furthermore, HMB increases the expression of brain glucose transporters 1 (GLUT1) and 3 (GLUT3), and mTOR phosphorylation, which translates in a higher protein synthesis in Neuro2a cells. Furthermore, Torin1 and rapamycin effects on MEF2 transcriptional activity and HMB-dependent neurite outgrowth support that HMB acts through mTORC2. Together, these findings provide clear evidence to support an important role of HMB in neurite outgrowth.

β-Hydroxy-β-Methylbutyrate (HMB) Promotes Neurite Outgrowth in Neuro2a Cells

PubMed Central

Girón, María D.; Cabrera, Elena; Campos, Nefertiti; Manzano, Manuel; Rueda, Ricardo; López-Pedrosa, Jose M.

2015-01-01

β-Hydroxy-β-methylbutyrate (HMB) has been shown to enhance cell survival, differentiation and protein turnover in muscle, mainly activating phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) and mitogen-activated protein kinases/ extracellular-signal-regulated kinases (MAPK/ERK) signaling pathways. Since these two pathways are related to neuronal survival and differentiation, in this study, we have investigated the neurotrophic effects of HMB in mouse neuroblastoma Neuro2a cells. In Neuro2a cells, HMB promotes differentiation to neurites independent from any effects on proliferation. These effects are mediated by activation of both the PI3K/Akt and the extracellular-signal-regulated kinases (ERK1/2) signaling as demonstrated by the use of specific inhibitors of these two pathways. As myocyte-enhancer factor 2 (MEF2) family of transcription factors are involved in neuronal survival and plasticity, the transcriptional activity and protein levels of MEF2 were also evaluated. HMB promoted MEF2-dependent transcriptional activity mediated by the activation of Akt and ERK1/2 pathways. Furthermore, HMB increases the expression of brain glucose transporters 1 (GLUT1) and 3 (GLUT3), and mTOR phosphorylation, which translates in a higher protein synthesis in Neuro2a cells. Furthermore, Torin1 and rapamycin effects on MEF2 transcriptional activity and HMB-dependent neurite outgrowth support that HMB acts through mTORC2. Together, these findings provide clear evidence to support an important role of HMB in neurite outgrowth. PMID:26267903

Regulation of the PI3K pathway through a p85α monomer–homodimer equilibrium | Office of Cancer Genomics

Cancer.gov

The canonical action of the p85α regulatory subunit of phosphatidylinositol 3-kinase (PI3K) is to associate with the p110α catalytic subunit to allow stimuli-dependent activation of the PI3K pathway. We elucidate a p110α-independent role of homodimerized p85α in the positive regulation of PTEN stability and activity.

The PINK1-PARKIN Mitochondrial Ubiquitylation Pathway Drives a Program of OPTN/NDP52 Recruitment and TBK1 Activation to Promote Mitophagy.

PubMed

Heo, Jin-Mi; Ordureau, Alban; Paulo, Joao A; Rinehart, Jesse; Harper, J Wade

2015-10-01

Damaged mitochondria are detrimental to cellular homeostasis. One mechanism for removal of damaged mitochondria involves the PINK1-PARKIN pathway, which poly-ubiquitylates damaged mitochondria to promote mitophagy. We report that assembly of ubiquitin chains on mitochondria triggers autophagy adaptor recruitment concomitantly with activation of the TBK1 kinase, which physically associates with OPTN, NDP52, and SQSTM1. TBK1 activation in HeLa cells requires OPTN and NDP52 and OPTN ubiquitin chain binding. In addition to the known role of S177 phosphorylation in OPTN on ATG8 recruitment, TBK1-dependent phosphorylation on S473 and S513 promotes ubiquitin chain binding in vitro as well as TBK1 activation, OPTN mitochondrial retention, and efficient mitophagy in vivo. These data reveal a self-reinforcing positive feedback mechanism that coordinates TBK1-dependent autophagy adaptor phosphorylation with the assembly of ubiquitin chains on mitochondria to facilitate efficient mitophagy, and mechanistically links genes mutated in Parkinson's disease and amyotrophic lateral sclerosis in a common selective autophagy pathway. Copyright © 2015 Elsevier Inc. All rights reserved.

Tivantinib (ARQ-197) exhibits anti-tumor activity with down-regulation of FAK in oral squamous cell carcinoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xi, Wei-Hong; Yang, Li-Yun; Cao, Zhong-Yi, E-mail: m18070383032@163.com

Oral squamous cell carcinoma (OSCC) is one of the most common cancers worldwide and the 5 years survival rate of the patients is about 60% in the USA, due to acquired chemotherapeutic resistance and metastasis of the disease. In this study, we found that tivantinib, a selective MET inhibitor, suppresses OCSS cell proliferation and colony formation, however, anti-tumor activities induced by tivantinib are independent of the inhibition of MET signaling pathway. In addition, tivantinib cause G2/M cell cycle arrest and caspases-dependent apoptosis in OSCC cell lines. We also found that tivantinib dose-dependently suppressed the activation and expression of FAK. Inmore » all, these data suggested that tivantinib may be developed as a chemotherapeutic agent to effectively treat certain cancers including OSCC. - Highlights: • Tivantinib suppresses OSCC cell growth independent of the inhibition of HGF/MET signaling pathway. • Tivantinib blocks cell cycle and induces caspases-mediated apoptosis. • Tivantinib elicits its anti-tumor activity with the inhibition of FAK signaling pathway.« less

HIV-1 Activates T Cell Signaling Independently of Antigen to Drive Viral Spread.

PubMed

Len, Alice C L; Starling, Shimona; Shivkumar, Maitreyi; Jolly, Clare

2017-01-24

HIV-1 spreads between CD4 T cells most efficiently through virus-induced cell-cell contacts. To test whether this process potentiates viral spread by activating signaling pathways, we developed an approach to analyze the phosphoproteome in infected and uninfected mixed-population T cells using differential metabolic labeling and mass spectrometry. We discovered HIV-1-induced activation of signaling networks during viral spread encompassing over 200 cellular proteins. Strikingly, pathways downstream of the T cell receptor were the most significantly activated, despite the absence of canonical antigen-dependent stimulation. The importance of this pathway was demonstrated by the depletion of proteins, and we show that HIV-1 Env-mediated cell-cell contact, the T cell receptor, and the Src kinase Lck were essential for signaling-dependent enhancement of viral dissemination. This study demonstrates that manipulation of signaling at immune cell contacts by HIV-1 is essential for promoting virus replication and defines a paradigm for antigen-independent T cell signaling. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Serine- and Threonine/Valine-Dependent Activation of PDK and Tor Orthologs Converge on Sch9 to Promote Aging

PubMed Central

Fabrizio, Paola; Wei, Min; Hu, Jia; Longo, Valter D.

2014-01-01

Dietary restriction extends longevity in organisms ranging from bacteria to mice and protects primates from a variety of diseases, but the contribution of each dietary component to aging is poorly understood. Here we demonstrate that glucose and specific amino acids promote stress sensitization and aging through the differential activation of the Ras/cAMP/PKA, PKH1/2 and Tor/S6K pathways. Whereas glucose sensitized cells through a Ras-dependent mechanism, threonine and valine promoted cellular sensitization and aging primarily by activating the Tor/S6K pathway and serine promoted sensitization via PDK1 orthologs Pkh1/2. Serine, threonine and valine activated a signaling network in which Sch9 integrates TORC1 and Pkh signaling via phosphorylation of threonines 570 and 737 and promoted intracellular relocalization and transcriptional inhibition of the stress resistance protein kinase Rim15. Because of the conserved pro-aging role of nutrient and growth signaling pathways in higher eukaryotes, these results raise the possibility that similar mechanisms contribute to aging in mammals. PMID:24516402

Concurrent Activation of Striatal Direct and Indirect Pathways During Action Initiation

PubMed Central

Cui, Guohong; Jun, Sang Beom; Jin, Xin; Pham, Michael D.

2014-01-01

Summary The basal ganglia are subcortical nuclei that control voluntary actions, and are affected by a number of debilitating neurological disorders1–4. The prevailing model of basal ganglia function proposes that two orthogonal projection circuits originating from distinct populations of spiny projection neurons (SPNs) in the striatum5,6 - the so-called direct and indirect pathways - have opposing effects on movement: while activity of direct-pathway SPNs purportedly facilitates movement, activity of indirect-pathway SPNs inhibits movement1,2. This model has been difficult to test due to the lack of methods to selectively measure the activity of direct- and indirect-pathway SPNs in freely moving animals. We developed a novel in-vivo method that allowed us to specifically measure direct- and indirect-pathway SPN activity using Cre-dependent viral expression of the genetically encoded calcium indicator (GECI) GCAMP3 in the dorsal striatum of D1-Cre (direct-pathway specific6,7) and A2A-Cre (indirect-pathway specific8,9) mice10. Using fiber optics and time-correlated single photon counting (TCSPC) in mice performing an operant task, we observed transient increases in neural activity in both direct- and indirect-pathway SPNs when animals initiated actions, but not when they were inactive. Concurrent activation of SPNs from both pathways in one hemisphere preceded the initiation of contraversive movements, and predicted the occurrence of specific movements within 500 ms. These observations challenge the classical view of basal ganglia function, and may have implications for understanding the origin of motor symptoms in basal ganglia disorders. PMID:23354054

The CSR-1 endogenous RNAi pathway ensures accurate transcriptional reprogramming during the oocyte-to-embryo transition in Caenorhabditis elegans.

PubMed

Fassnacht, Christina; Tocchini, Cristina; Kumari, Pooja; Gaidatzis, Dimos; Stadler, Michael B; Ciosk, Rafal

2018-03-01

Endogenous RNAi (endoRNAi) is a conserved mechanism for fine-tuning gene expression. In the nematode Caenorhabditis elegans, several endoRNAi pathways are required for the successful development of reproductive cells. The CSR-1 endoRNAi pathway promotes germ cell development, primarily by facilitating the expression of germline genes. In this study, we report a novel function for the CSR-1 pathway in preventing premature activation of embryonic transcription in the developing oocytes, which is accompanied by a general Pol II activation. This CSR-1 function requires its RNase activity, suggesting that, by controlling the levels of maternal mRNAs, CSR-1-dependent endoRNAi contributes to an orderly reprogramming of transcription during the oocyte-to-embryo transition.

The CSR-1 endogenous RNAi pathway ensures accurate transcriptional reprogramming during the oocyte-to-embryo transition in Caenorhabditis elegans

PubMed Central

Tocchini, Cristina; Kumari, Pooja; Gaidatzis, Dimos

2018-01-01

Endogenous RNAi (endoRNAi) is a conserved mechanism for fine-tuning gene expression. In the nematode Caenorhabditis elegans, several endoRNAi pathways are required for the successful development of reproductive cells. The CSR-1 endoRNAi pathway promotes germ cell development, primarily by facilitating the expression of germline genes. In this study, we report a novel function for the CSR-1 pathway in preventing premature activation of embryonic transcription in the developing oocytes, which is accompanied by a general Pol II activation. This CSR-1 function requires its RNase activity, suggesting that, by controlling the levels of maternal mRNAs, CSR-1-dependent endoRNAi contributes to an orderly reprogramming of transcription during the oocyte-to-embryo transition. PMID:29579041

Arsenic trioxide mediates HAPI microglia inflammatory response and subsequent neuron apoptosis through p38/JNK MAPK/STAT3 pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mao, Jiamin

Arsenic is a widely distributed toxic metalloid all over the world. Inorganic arsenic species are supposed to affect astrocytic functions and to cause neuron apoptosis in CNS. Microglias are the key cell type involved in innate immune responses in CNS, and microglia activation has been linked to inflammation and neurotoxicity. In this study, using ELISA, we showed that Arsenic trioxide up-regulated the expression and secretion of IL-1β in a dose-dependent manner and a time-dependent manner in cultured HAPI microglia cells. The secretion of IL-1β caused the apoptosis of SH-SY5Y. These pro-inflammatory responses were inhibited by the STAT3 blocker, AG490 andmore » P38/JNK MAPK blockers SB202190, SP600125. Further, Arsenic trioxide exposure could induce phosphorylation and activation of STAT3, and the translocation of STAT3 from the cytosol to the nucleus in this HAPI microglia cell line. Thus, the STAT3 signaling pathway can be activated after Arsenic trioxide treatment. However, P38/JNK MAPK blockers SB202190, SP600125 also obviously attenuated STAT3 activation and transnuclear transport induced by Arsenic trioxide. In concert with these results, we highlighted that the secretion of IL-1β and STAT3 activation induced by Arsenic trioxide can be mediated by elevation of P38/JNK MAPK in HAPI microglia cells and then induced the toxicity of neurons. - Highlights: • Arsenic trioxide exposure induced expression of IL-β in HAPI microglia. • Arsenic trioxide exposure induced activation of MAPK pathways in HAPI microglia. • Arsenic trioxide exposure induced activation of STAT3 pathways in HAPI microglia. • The expression of IL-β though P38/JNK MAPK/STAT3 pathways in HAPI microglia.« less

Computation of restoration of ligand response in the random kinetics of a prostate cancer cell signaling pathway.

PubMed

Dana, Saswati; Nakakuki, Takashi; Hatakeyama, Mariko; Kimura, Shuhei; Raha, Soumyendu

2011-01-01

Mutation and/or dysfunction of signaling proteins in the mitogen activated protein kinase (MAPK) signal transduction pathway are frequently observed in various kinds of human cancer. Consistent with this fact, in the present study, we experimentally observe that the epidermal growth factor (EGF) induced activation profile of MAP kinase signaling is not straightforward dose-dependent in the PC3 prostate cancer cells. To find out what parameters and reactions in the pathway are involved in this departure from the normal dose-dependency, a model-based pathway analysis is performed. The pathway is mathematically modeled with 28 rate equations yielding those many ordinary differential equations (ODE) with kinetic rate constants that have been reported to take random values in the existing literature. This has led to us treating the ODE model of the pathways kinetics as a random differential equations (RDE) system in which the parameters are random variables. We show that our RDE model captures the uncertainty in the kinetic rate constants as seen in the behavior of the experimental data and more importantly, upon simulation, exhibits the abnormal EGF dose-dependency of the activation profile of MAP kinase signaling in PC3 prostate cancer cells. The most likely set of values of the kinetic rate constants obtained from fitting the RDE model into the experimental data is then used in a direct transcription based dynamic optimization method for computing the changes needed in these kinetic rate constant values for the restoration of the normal EGF dose response. The last computation identifies the parameters, i.e., the kinetic rate constants in the RDE model, that are the most sensitive to the change in the EGF dose response behavior in the PC3 prostate cancer cells. The reactions in which these most sensitive parameters participate emerge as candidate drug targets on the signaling pathway. 2011 Elsevier Ireland Ltd. All rights reserved.

Mechanisms of dopamine D1 receptor-mediated ERK1/2 activation in the parkinsonian striatum and their modulation by metabotropic glutamate receptor type 5.

PubMed

Fieblinger, Tim; Sebastianutto, Irene; Alcacer, Cristina; Bimpisidis, Zisis; Maslava, Natallia; Sandberg, Sabina; Engblom, David; Cenci, M Angela

2014-03-26

In animal models of Parkinson's disease, striatal overactivation of ERK1/2 via dopamine (DA) D1 receptors is the hallmark of a supersensitive molecular response associated with dyskinetic behaviors. Here we investigate the pathways involved in D1 receptor-dependent ERK1/2 activation using acute striatal slices from rodents with unilateral 6-hydroxydopamine (6-OHDA) lesions. Application of the dopamine D1-like receptor agonist SKF38393 induced ERK1/2 phosphorylation and downstream signaling in the DA-denervated but not the intact striatum. This response was mediated through a canonical D1R/PKA/MEK1/2 pathway and independent of ionotropic glutamate receptors but blocked by antagonists of L-type calcium channels. Coapplication of an antagonist of metabotropic glutamate receptor type 5 (mGluR5) or its downstream signaling molecules (PLC, PKC, IP3 receptors) markedly attenuated SKF38393-induced ERK1/2 activation. The role of striatal mGluR5 in D1-dependent ERK1/2 activation was confirmed in vivo in 6-OHDA-lesioned animals treated systemically with SKF38393. In one experiment, local infusion of the mGluR5 antagonist MTEP in the DA-denervated rat striatum attenuated the activation of ERK1/2 signaling by SKF38393. In another experiment, 6-OHDA lesions were applied to transgenic mice with a cell-specific knockdown of mGluR5 in D1 receptor-expressing neurons. These mice showed a blunted striatal ERK1/2 activation in response to SFK38393 treatment. Our results reveal that D1-dependent ERK1/2 activation in the DA-denervated striatum depends on a complex interaction between PKA- and Ca(2+)-dependent signaling pathways that is critically modulated by striatal mGluR5.

Paraoxon induces apoptosis in EL4 cells via activation of mitochondrial pathways.

PubMed

Saleh, A M; Vijayasarathy, C; Masoud, L; Kumar, L; Shahin, A; Kambal, A

2003-07-01

The toxicity of organophosphorus compounds, such as paraoxon (POX), is due to their anticholinesterase action. Recently, we have shown that, at noncholinergic doses (1 to 10 nM), POX (the bioactive metabolite of parathion) causes apoptotic cell death in murine EL4 T-lymphocytic leukemia cell line through activation of caspase-3. In this study, by employing caspase-specific inhibitors, we extend our observations to elucidate the sequence of events involved in POX-stimulated apoptosis. Pretreatment of EL4 cells with the caspase-9-specific inhibitor zLEHD-fmk attenuated POX-induced apoptosis in a dose-dependent manner, whereas the caspase-8 inhibitor zIETD-fmk had no effect. Furthermore, the activation of caspase-9, -8, and -3 in response to POX treatment was completely inhibited in the presence of zLEHD-fmk, implicating the involvement of caspase 9-dependent mitochondrial pathways in POX-stimulated apoptosis. Indeed, under both in vitro and in vivo conditions, POX triggered a dose- and time-dependent translocation of cytochrome c from mitochondria into the cytosol, as assessed by Western blot analysis. Investigation of the mechanism of cytochrome c release revealed that POX disrupted mitochondrial transmembrane potential. Neither this effect nor cytchrome c release was dependent on caspase activation, since the general inhibitor of the caspase family zVAD-fmk did not influence both processes. Finally, POX treatment also resulted in a time-dependent up-regulation and translocation of the proapoptotic molecule Bax to mitochondria. Inhibition of this event by zVAD-fmk suggests that the activation and translocation of Bax to mitochondria is subsequent to activation of the caspase cascades. The results indicate that POX induces apoptosis in EL4 cells through a direct effect on mitochondria by disrupting its transmembrane potential, causing the release of cytochrome c into the cytosol and subsequent activation of caspase-9. Inhibition of this specific pathway might provide a useful strategy to minimize organophosphate-induced poisoning.

Leucine facilitates insulin signaling through a Gαi protein-dependent signaling pathway in hepatocytes.

PubMed

Yang, Xuefeng; Mei, Shuang; Wang, Xiaolei; Li, Xiang; Liu, Rui; Ma, Yan; Hao, Liping; Yao, Ping; Liu, Liegang; Sun, Xiufa; Gu, Haihua; Liu, Zhenqi; Cao, Wenhong

2013-03-29

In this study, we addressed the direct effect of leucine on insulin signaling. In investigating the associated mechanisms, we found that leucine itself does not activate the classical Akt- or ERK1/2 MAP kinase-dependent signaling pathways but can facilitate the insulin-induced phosphorylations of Akt(473) and ERK1/2 in a time- and dose-dependent manner in cultured hepatocytes. The leucine-facilitated insulin-induced phosphorylation of Akt at residue 473 was not affected by knocking down the key component of mTORC1 or -2 complexes but was blocked by inhibition of c-Src (PP2), PI3K (LY294002), Gαi protein (pertussis toxin or siRNA against Gαi1 gene, or β-arrestin 2 (siRNA)). Similarly, the leucine-facilitated insulin activation of ERK1/2 was also blunted by pertussis toxin. We further show that leucine facilitated the insulin-mediated suppression of glucose production and expression of key gluconeogenic genes in a Gαi1 protein-dependent manner in cultured primary hepatocytes. Together, these results show that leucine can directly facilitate insulin signaling through a Gαi protein-dependent intracellular signaling pathway. This is the first evidence showing that macronutrients like amino acid leucine can facilitate insulin signaling through G proteins directly.

Leucine Facilitates Insulin Signaling through a Gαi Protein-dependent Signaling Pathway in Hepatocytes*

PubMed Central

Yang, Xuefeng; Mei, Shuang; Wang, Xiaolei; Li, Xiang; Liu, Rui; Ma, Yan; Hao, Liping; Yao, Ping; Liu, Liegang; Sun, Xiufa; Gu, Haihua; Liu, Zhenqi; Cao, Wenhong

2013-01-01

In this study, we addressed the direct effect of leucine on insulin signaling. In investigating the associated mechanisms, we found that leucine itself does not activate the classical Akt- or ERK1/2 MAP kinase-dependent signaling pathways but can facilitate the insulin-induced phosphorylations of Akt473 and ERK1/2 in a time- and dose-dependent manner in cultured hepatocytes. The leucine-facilitated insulin-induced phosphorylation of Akt at residue 473 was not affected by knocking down the key component of mTORC1 or -2 complexes but was blocked by inhibition of c-Src (PP2), PI3K (LY294002), Gαi protein (pertussis toxin or siRNA against Gαi1 gene, or β-arrestin 2 (siRNA)). Similarly, the leucine-facilitated insulin activation of ERK1/2 was also blunted by pertussis toxin. We further show that leucine facilitated the insulin-mediated suppression of glucose production and expression of key gluconeogenic genes in a Gαi1 protein-dependent manner in cultured primary hepatocytes. Together, these results show that leucine can directly facilitate insulin signaling through a Gαi protein-dependent intracellular signaling pathway. This is the first evidence showing that macronutrients like amino acid leucine can facilitate insulin signaling through G proteins directly. PMID:23404499

Cyclic lipopeptide iturin A structure-dependently induces defense response in Arabidopsis plants by activating SA and JA signaling pathways.

PubMed

Kawagoe, Yumi; Shiraishi, Soma; Kondo, Hiroko; Yamamoto, Shoko; Aoki, Yoshinao; Suzuki, Shunji

2015-05-15

Iturin A is the most well studied antifungal cyclic lipopeptide produced by Bacillus species that are frequently utilized as biological control agents. Iturin A not only shows strong antifungal activity against phytopathogens but also induces defense response in plants, thereby reducing plant disease severity. Here we report the defense signaling pathways triggered by iturin A in Arabidopsis salicylic acid (SA) or jasmonic acid (JA)-insensitive mutants. Iturin A activated the transcription of defense genes PR1 and PDF1.2 through the SA and JA signaling pathways, respectively. The role of iturin A as an elicitor was dependent on the cyclization of the seven amino acids and/or the β-hydroxy fatty acid chain. The iturin A derivative peptide, NH2-(L-Asn)-(D-Tyr)-(D-Asn)-(L-Gln)-(L-Pro)-(D-Asn)-(L-Ser)-COOH, completely suppressed PR1 and PDF1.2 gene expression in wild Arabidopsis plants. The identification of target molecules binding to iturin A and its derivative peptide is expected to shed new light on defense response in plants through the SA and JA signaling pathways. Copyright © 2015 Elsevier Inc. All rights reserved.

FGF1 protects neuroblastoma SH-SY5Y cells from p53-dependent apoptosis through an intracrine pathway regulated by FGF1 phosphorylation

PubMed Central

Pirou, Caroline; Montazer-Torbati, Fatemeh; Jah, Nadège; Delmas, Elisabeth; Lasbleiz, Christelle; Mignotte, Bernard; Renaud, Flore

2017-01-01

Neuroblastoma, a sympathetic nervous system tumor, accounts for 15% of cancer deaths in children. In contrast to most human tumors, p53 is rarely mutated in human primary neuroblastoma, suggesting impaired p53 activation in neuroblastoma. Various studies have shown correlations between fgf1 expression levels and both prognosis severity and tumor chemoresistance. As we previously showed that fibroblast growth factor 1 (FGF1) inhibited p53-dependent apoptosis in neuron-like PC12 cells, we initiated the study of the interaction between the FGF1 and p53 pathways in neuroblastoma. We focused on the activity of either extracellular FGF1 by adding recombinant rFGF1 in media, or of intracellular FGF1 by overexpression in human SH-SY5Y and mouse N2a neuroblastoma cell lines. In both cell lines, the genotoxic drug etoposide induced a classical mitochondrial p53-dependent apoptosis. FGF1 was able to inhibit p53-dependent apoptosis upstream of mitochondrial events in SH-SY5Y cells by both extracellular and intracellular pathways. Both rFGF1 addition and etoposide treatment increased fgf1 expression in SH-SY5Y cells. Conversely, rFGF1 or overexpressed FGF1 had no effect on p53-dependent apoptosis and fgf1 expression in neuroblastoma N2a cells. Using different FGF1 mutants (that is, FGF1K132E, FGF1S130A and FGF1S130D), we further showed that the C-terminal domain and phosphorylation of FGF1 regulate its intracrine anti-apoptotic activity in neuroblastoma SH-SY5Y cells. This study provides the first evidence for a role of an intracrine growth factor pathway on p53-dependent apoptosis in neuroblastoma, and could lead to the identification of key regulators involved in neuroblastoma tumor progression and chemoresistance. PMID:29048426

IFNβ/TNFα synergism induces a non-canonical STAT2/IRF9-dependent pathway triggering a novel DUOX2 NADPH Oxidase-mediated airway antiviral response

PubMed Central

Fink, Karin; Martin, Lydie; Mukawera, Esperance; Chartier, Stéfany; De Deken, Xavier; Brochiero, Emmanuelle; Miot, Françoise; Grandvaux, Nathalie

2013-01-01

Airway epithelial cells are key initial innate immune responders in the fight against respiratory viruses, primarily via the secretion of antiviral and proinflammatory cytokines that act in an autocrine/paracrine fashion to trigger the establishment of an antiviral state. It is currently thought that the early antiviral state in airway epithelial cells primarily relies on IFNβ secretion and the subsequent activation of the interferon-stimulated gene factor 3 (ISGF3) transcription factor complex, composed of STAT1, STAT2 and IRF9, which regulates the expression of a panoply of interferon-stimulated genes encoding proteins with antiviral activities. However, the specific pathways engaged by the synergistic action of different cytokines during viral infections, and the resulting physiological outcomes are still ill-defined. Here, we unveil a novel delayed antiviral response in the airways, which is initiated by the synergistic autocrine/paracrine action of IFNβ and TNFα, and signals through a non-canonical STAT2- and IRF9-dependent, but STAT1-independent cascade. This pathway ultimately leads to the late induction of the DUOX2 NADPH oxidase expression. Importantly, our study uncovers that the development of the antiviral state relies on DUOX2-dependent H2O2 production. Key antiviral pathways are often targeted by evasion strategies evolved by various pathogenic viruses. In this regard, the importance of the novel DUOX2-dependent antiviral pathway is further underlined by the observation that the human respiratory syncytial virus is able to subvert DUOX2 induction. PMID:23545780

Jagged1 is the pathological link between Wnt and Notch pathways in colorectal cancer

PubMed Central

Rodilla, Verónica; Villanueva, Alberto; Obrador-Hevia, Antonia; Robert-Moreno, Àlex; Fernández-Majada, Vanessa; Grilli, Andrea; López-Bigas, Nuria; Bellora, Nicolás; Albà, M. Mar; Torres, Ferran; Duñach, Mireia; Sanjuan, Xavier; Gonzalez, Sara; Gridley, Thomas; Capella, Gabriel; Bigas, Anna; Espinosa, Lluís

2009-01-01

Notch has been linked to β-catenin-dependent tumorigenesis; however, the mechanisms leading to Notch activation and the contribution of the Notch pathway to colorectal cancer is not yet understood. By microarray analysis, we have identified a group of genes downstream of Wnt/β-catenin (down-regulated when blocking Wnt/β-catenin) that are directly regulated by Notch (repressed by γ-secretase inhibitors and up-regulated by active Notch1 in the absence of β-catenin signaling). We demonstrate that Notch is downstream of Wnt in colorectal cancer cells through β-catenin-mediated transcriptional activation of the Notch-ligand Jagged1. Consistently, expression of activated Notch1 partially reverts the effects of blocking Wnt/β-catenin pathway in tumors implanted s.c. in nude mice. Crossing APCMin/+ with Jagged1+/Δ mice is sufficient to significantly reduce the size of the polyps arising in the APC mutant background indicating that Notch is an essential modulator of tumorigenesis induced by nuclear β-catenin. We show that this mechanism is operating in human tumors from Familial Adenomatous Polyposis patients. We conclude that Notch activation, accomplished by β-catenin-mediated up-regulation of Jagged1, is required for tumorigenesis in the intestine. The Notch-specific genetic signature is sufficient to block differentiation and promote vasculogenesis in tumors whereas proliferation depends on both pathways. PMID:19325125

Jagged1 is the pathological link between Wnt and Notch pathways in colorectal cancer.

PubMed

Rodilla, Verónica; Villanueva, Alberto; Obrador-Hevia, Antonia; Robert-Moreno, Alex; Fernández-Majada, Vanessa; Grilli, Andrea; López-Bigas, Nuria; Bellora, Nicolás; Albà, M Mar; Torres, Ferran; Duñach, Mireia; Sanjuan, Xavier; Gonzalez, Sara; Gridley, Thomas; Capella, Gabriel; Bigas, Anna; Espinosa, Lluís

2009-04-14

Notch has been linked to beta-catenin-dependent tumorigenesis; however, the mechanisms leading to Notch activation and the contribution of the Notch pathway to colorectal cancer is not yet understood. By microarray analysis, we have identified a group of genes downstream of Wnt/beta-catenin (down-regulated when blocking Wnt/beta-catenin) that are directly regulated by Notch (repressed by gamma-secretase inhibitors and up-regulated by active Notch1 in the absence of beta-catenin signaling). We demonstrate that Notch is downstream of Wnt in colorectal cancer cells through beta-catenin-mediated transcriptional activation of the Notch-ligand Jagged1. Consistently, expression of activated Notch1 partially reverts the effects of blocking Wnt/beta-catenin pathway in tumors implanted s.c. in nude mice. Crossing APC(Min/+) with Jagged1(+/Delta) mice is sufficient to significantly reduce the size of the polyps arising in the APC mutant background indicating that Notch is an essential modulator of tumorigenesis induced by nuclear beta-catenin. We show that this mechanism is operating in human tumors from Familial Adenomatous Polyposis patients. We conclude that Notch activation, accomplished by beta-catenin-mediated up-regulation of Jagged1, is required for tumorigenesis in the intestine. The Notch-specific genetic signature is sufficient to block differentiation and promote vasculogenesis in tumors whereas proliferation depends on both pathways.

Arctigenin induces apoptosis in colon cancer cells through ROS/p38MAPK pathway.

PubMed

Li, Qing-chun; Liang, Yun; Tian, Yuan; Hu, Guang-rui

2016-01-01

In the current study the antiproliferative effect of arctigenin, plant lignin, was evaluated on human colon cancer cell line HT-29. Furthermore, attempts were made to explore the signaling mechanism which may be responsible for its effect. Cell growth inhibition was assessed by MTT and LDH assays. Flow cytometric analysis was performed to determine cell arrest in the cell cycle phase and apoptosis. Furthermore, to confirm the apoptotic activity of arctigenin, caspase-9 and -3 activities analysis was performed. The levels of reactive oxygen species (ROS) and p38 mitogen activated protein kinase (MAPK) were investigated to determine their role in inducing apoptosis in arctigenin-treated HT-29 colon cancer cell line. MTT and LDH results demonstrated significant cell growth inhibitory effect of arctigenin on HT-29 cells in a dose-dependent manner. Furthermore, increase in cell number arrested at G2/M phase was observed in flow cytometric analysis upon arctigenin treatment. In addition, arctigenin increased the apoptotic ratio in a dose-dependent manner. The involvement of intrinsic apoptotic pathway was indicated by the activation of caspase-9 and -3. Moreover, increased ROS production, activation of p38 MAPK and changes in mitochondrial membrane potential (ΔΨm) also revealed the role of intrinsic apoptotic signaling pathway in cell growth inhibition after arctigenin exposure. Arctigenin induces apoptosis in HT-29 colon cancer cells by regulating ROS and p38 MAPK pathways.

Mechanism of interleukin-13 production by granulocyte-macrophage colony-stimulating factor-dependent macrophages via protease-activated receptor-2.

PubMed

Yamaguchi, Rui; Yamamoto, Takatoshi; Sakamoto, Arisa; Ishimaru, Yasuji; Narahara, Shinji; Sugiuchi, Hiroyuki; Hirose, Eiji; Yamaguchi, Yasuo

2015-06-01

Granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes classically activated M1 macrophages. GM-CSF upregulates protease-activated receptor-2 (PAR-2) protein expression and activation of PAR-2 by human neutrophil elastase (HNE) regulates cytokine production. This study investigated the mechanism of PAR-2-mediated interleukin (IL)-13 production by GM-CSF-dependent macrophages stimulated with HNE. Adherent macrophages were obtained from primary cultures of human mononuclear cells. After stimulation with HNE to activate the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway, IL-13 mRNA and protein levels were assessed by the reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. PAR-2 protein was detected in GM-CSF-dependent macrophages by Western blotting. Unexpectedly, PD98059 (an ERK1 inhibitor) increased IL-13 production, even at higher concentrations. Interestingly, U0126 (an ERK1/2 inhibitor) reduced IL-13 production in a concentration-dependent manner. Neither SB203580 (a p38alpha/p38beta inhibitor) nor BIRB796 (a p38gamma/p38delta inhibitor) affected IL-13 production, while TMB-8 (a calcium chelator) diminished IL-13 production. Stimulation with HNE promoted the production of IL-13 (a Th2 cytokine) by GM-CSF-dependent M1 macrophages. PAR-2-mediated IL-13 production may be dependent on the Ca(2+)/ERK2 signaling pathway. Copyright © 2015 Elsevier Inc. All rights reserved.

AKAP-Lbc mobilizes a cardiac hypertrophy signaling pathway.

PubMed

Carnegie, Graeme K; Soughayer, Joseph; Smith, F Donelson; Pedroja, Benjamin S; Zhang, Fang; Diviani, Dario; Bristow, Michael R; Kunkel, Maya T; Newton, Alexandra C; Langeberg, Lorene K; Scott, John D

2008-10-24

Elevated catecholamines in the heart evoke transcriptional activation of the Myocyte Enhancer Factor (MEF) pathway to induce a cellular response known as pathological myocardial hypertrophy. We have discovered that the A-Kinase Anchoring Protein (AKAP)-Lbc is upregulated in hypertrophic cardiomyocytes. It coordinates activation and movement of signaling proteins that initiate MEF2-mediated transcriptional reprogramming events. Live-cell imaging, fluorescent kinase activity reporters, and RNA interference techniques show that AKAP-Lbc couples activation of protein kinase D (PKD) with the phosphorylation-dependent nuclear export of the class II histone deacetylase HDAC5. These studies uncover a role for AKAP-Lbc in which increased expression of the anchoring protein selectively amplifies a signaling pathway that drives cardiac myocytes toward a pathophysiological outcome.

Recruitment of Additional Corticospinal Pathways in the Human Brain with State-Dependent Paired Associative Stimulation.

PubMed

Kraus, Dominic; Naros, Georgios; Guggenberger, Robert; Leão, Maria Teresa; Ziemann, Ulf; Gharabaghi, Alireza

2018-02-07

Standard brain stimulation protocols modify human motor cortex excitability by modulating the gain of the activated corticospinal pathways. However, the restoration of motor function following lesions of the corticospinal tract requires also the recruitment of additional neurons to increase the net corticospinal output. For this purpose, we investigated a novel protocol based on brain state-dependent paired associative stimulation.Motor imagery (MI)-related electroencephalography was recorded in healthy males and females for brain state-dependent control of both cortical and peripheral stimulation in a brain-machine interface environment. State-dependency was investigated with concurrent, delayed, and independent stimulation relative to the MI task. Specifically, sensorimotor event-related desynchronization (ERD) in the β-band (16-22 Hz) triggered peripheral stimulation through passive hand opening by a robotic orthosis and transcranial magnetic stimulation to the respective cortical motor representation, either synchronously or subsequently. These MI-related paradigms were compared with paired cortical and peripheral input applied independent of the brain state. Cortical stimulation resulted in a significant increase in corticospinal excitability only when applied brain state-dependently and synchronously to peripheral input. These gains were resistant to a depotentiation task, revealed a nonlinear evolution of plasticity, and were mediated via the recruitment of additional corticospinal neurons rather than via synchronization of neuronal firing. Recruitment of additional corticospinal pathways may be achieved when cortical and peripheral inputs are applied concurrently, and during β-ERD. These findings resemble a gating mechanism and are potentially important for developing closed-loop brain stimulation for the treatment of hand paralysis following lesions of the corticospinal tract. SIGNIFICANCE STATEMENT The activity state of the motor system influences the excitability of corticospinal pathways to external input. State-dependent interventions harness this property to increase the connectivity between motor cortex and muscles. These stimulation protocols modulate the gain of the activated pathways, but not the overall corticospinal recruitment. In this study, a brain-machine interface paired peripheral stimulation through passive hand opening with transcranial magnetic stimulation to the respective cortical motor representation during volitional β-band desynchronization. Cortical stimulation resulted in the recruitment of additional corticospinal pathways, but only when applied brain state-dependently and synchronously to peripheral input. These effects resemble a gating mechanism and may be important for the restoration of motor function following lesions of the corticospinal tract. Copyright © 2018 the authors 0270-6474/18/381397-12$15.00/0.

Cordycepin induced eryptosis in mouse erythrocytes through a Ca2+-dependent pathway without caspase-3 activation.

PubMed

Lui, Julian C K; Wong, Judy W Y; Suen, Y K; Kwok, T T; Fung, K P; Kong, S K

2007-12-01

Cordyceps sinensis is a prized traditional Chinese medicine and its major component cordycepin is found to have anti-leukemia activities. However, its cytotoxicity in erythrocytes was unclear. To examine the effect of cordycepin on the induction of eryptosis (an apoptosis-like process in enucleated erythrocytes), flow cytometric assays based on membrane integrity and asymmetry were employed. For comparison, analyses were performed in parallel with two other anti-leukemia agents, indirubin 3'-monoxime (IDM) and As2O3. We found that at the IC50 against leukemia HL-60, cordycepin elicited eryptosis while IDM and As2O3 showed no erythrotoxicity in mouse erythrocytes. Mechanistically, cordycepin increased the [Ca2+]i and activated mu-calpain protease in a dose-dependent manner. Yet, no caspase-3 activation was observed in the cordycepin-treated erythrocytes. When extracellular Ca2+ was depleted, both the cordycepin-induced eryptosis and mu-calpain cleavage were suppressed. Our study therefore demonstrated for the first time that cordycepin induces eryptosis through a calcium-dependent pathway in the absence of mitochondria and caspase-3 activation.

Increases in cAMP, MAPK Activity and CREB Phosphorylation during REM Sleep: Implications for REM Sleep and Memory Consolidation

PubMed Central

Luo, Jie; Phan, Trongha X.; Yang, Yimei; Garelick, Michael G.; Storm, Daniel R.

2013-01-01

The cyclic adenosine monophosphate (cAMP), mitogen-activated protein kinase (MAPK) and cAMP response element-binding protein (CREB) transcriptional pathway is required for consolidation of hippocampus-dependent memory. In mice, this pathway undergoes a circadian oscillation required for memory persistence that reaches a peak during the daytime. Since mice exhibit polyphasic sleep patterns during the day, this suggested the interesting possibility that cAMP, MAPK activity and CREB phosphorylation may be elevated during sleep. Here, we report that cAMP, phospho-p44/42 MAPK and phospho-CREB are higher in rapid eye movement (REM) sleep compared to awake mice but are not elevated in non-rapid eye movement (NREM) sleep. This peak of activity during REM sleep does not occur in mice lacking calmodulin-stimulated adenylyl cyclases, a mouse strain that learns but cannot consolidate hippocampus-dependent memory. We conclude that a preferential increase in cAMP, MAPK activity and CREB phosphorylation during REM sleep may contribute to hippocampus-dependent memory consolidation. PMID:23575844

Redox regulation in cancer stem cells

USDA-ARS?s Scientific Manuscript database

Reactive oxygen species (ROS) and ROS-dependent (redox regulation) signaling pathways and transcriptional activities are thought to be critical in stem cell self-renewal and differentiation during growth and organogenesis. Aberrant ROS burst and dysregulation of those ROS-dependent cellular processe...

Disruption of the vacuolar calcium-ATPases in Arabidopsis results in the activation of a salicylic acid-dependent programmed cell death pathway.

PubMed

Boursiac, Yann; Lee, Sang Min; Romanowsky, Shawn; Blank, Robert; Sladek, Chris; Chung, Woo Sik; Harper, Jeffrey F

2010-11-01

Calcium (Ca(2+)) signals regulate many aspects of plant development, including a programmed cell death pathway that protects plants from pathogens (hypersensitive response). Cytosolic Ca(2+) signals result from a combined action of Ca(2+) influx through channels and Ca(2+) efflux through pumps and cotransporters. Plants utilize calmodulin-activated Ca(2+) pumps (autoinhibited Ca(2+)-ATPase [ACA]) at the plasma membrane, endoplasmic reticulum, and vacuole. Here, we show that a double knockout mutation of the vacuolar Ca(2+) pumps ACA4 and ACA11 in Arabidopsis (Arabidopsis thaliana) results in a high frequency of hypersensitive response-like lesions. The appearance of macrolesions could be suppressed by growing plants with increased levels (greater than 15 mm) of various anions, providing a method for conditional suppression. By removing plants from a conditional suppression, lesion initials were found to originate primarily in leaf mesophyll cells, as detected by aniline blue staining. Initiation and spread of lesions could also be suppressed by disrupting the production or accumulation of salicylic acid (SA), as shown by combining aca4/11 mutations with a sid 2 (for salicylic acid induction-deficient2) mutation or expression of the SA degradation enzyme NahG. This indicates that the loss of the vacuolar Ca(2+) pumps by itself does not cause a catastrophic defect in ion homeostasis but rather potentiates the activation of a SA-dependent programmed cell death pathway. Together, these results provide evidence linking the activity of the vacuolar Ca(2+) pumps to the control of a SA-dependent programmed cell death pathway in plants.

Aripiprazole and Haloperidol Activate GSK3β-Dependent Signalling Pathway Differentially in Various Brain Regions of Rats

PubMed Central

Pan, Bo; Huang, Xu-Feng; Deng, Chao

2016-01-01

Aripiprazole, a dopamine D2 receptor (D2R) partial agonist, possesses a unique clinical profile. Glycogen synthase kinase 3β (GSK3β)-dependent signalling pathways have been implicated in the pathophysiology of schizophrenia and antipsychotic drug actions. The present study examined whether aripiprazole differentially affects the GSK3β-dependent signalling pathways in the prefrontal cortex (PFC), nucleus accumbens (NAc), and caudate putamen (CPu), in comparison with haloperidol (a D2R antagonist) and bifeprunox (a D2R partial agonist). Rats were orally administrated aripiprazole (0.75 mg/kg), bifeprunox (0.8 mg/kg), haloperidol (0.1 mg/kg) or vehicle three times per day for one week. The levels of protein kinase B (Akt), p-Akt, GSK3β, p-GSK3β, dishevelled (Dvl)-3, and β-catenin were measured by Western Blots. Aripiprazole increased GSK3β phosphorylation in the PFC and NAc, respectively, while haloperidol elevated it in the NAc only. However, Akt activity was not changed by any of these drugs. Additionally, both aripiprazole and haloperidol, but not bifeprunox, increased the expression of Dvl-3 and β-catenin in the NAc. The present study suggests that activation of GSK3β phosphorylation in the PFC and NAc may be involved in the clinical profile of aripiprazole; additionally, aripiprazole can increase GSK3β phosphorylation via the Dvl-GSK3β-β-catenin signalling pathway in the NAc, probably due to its relatively low intrinsic activity at D2Rs. PMID:27043526

Loss of the Drosophila cell polarity regulator Scribbled promotes epithelial tissue overgrowth and cooperation with oncogenic Ras-Raf through impaired Hippo pathway signaling

PubMed Central

2011-01-01

Background Epithelial neoplasias are associated with alterations in cell polarity and excessive cell proliferation, yet how these neoplastic properties are related to one another is still poorly understood. The study of Drosophila genes that function as neoplastic tumor suppressors by regulating both of these properties has significant potential to clarify this relationship. Results Here we show in Drosophila that loss of Scribbled (Scrib), a cell polarity regulator and neoplastic tumor suppressor, results in impaired Hippo pathway signaling in the epithelial tissues of both the eye and wing imaginal disc. scrib mutant tissue overgrowth, but not the loss of cell polarity, is dependent upon defective Hippo signaling and can be rescued by knockdown of either the TEAD/TEF family transcription factor Scalloped or the transcriptional coactivator Yorkie in the eye disc, or reducing levels of Yorkie in the wing disc. Furthermore, loss of Scrib sensitizes tissue to transformation by oncogenic Ras-Raf signaling, and Yorkie-Scalloped activity is required to promote this cooperative tumor overgrowth. The inhibition of Hippo signaling in scrib mutant eye disc clones is not dependent upon JNK activity, but can be significantly rescued by reducing aPKC kinase activity, and ectopic aPKC activity is sufficient to impair Hippo signaling in the eye disc, even when JNK signaling is blocked. In contrast, warts mutant overgrowth does not require aPKC activity. Moreover, reducing endogenous levels of aPKC or increasing Scrib or Lethal giant larvae levels does not promote increased Hippo signaling, suggesting that aPKC activity is not normally rate limiting for Hippo pathway activity. Epistasis experiments suggest that Hippo pathway inhibition in scrib mutants occurs, at least in part, downstream or in parallel to both the Expanded and Fat arms of Hippo pathway regulation. Conclusions Loss of Scrib promotes Yorkie/Scalloped-dependent epithelial tissue overgrowth, and this is also important for driving cooperative tumor overgrowth with oncogenic Ras-Raf signaling. Whether this is also the case in human cancers now warrants investigation since the cell polarity function of Scrib and its capacity to restrain oncogene-mediated transformation, as well as the tissue growth control function of the Hippo pathway, are conserved in mammals. PMID:21955824

Activation of peroxisome proliferator-activated receptors (PPARs) by their ligands and protein kinase A activators

PubMed Central

Lazennec, Gwendal; Canaple, Laurence; Saugy, Damien; Wahli, Walter

2000-01-01

The nuclear peroxisome proliferator-activated receptors (PPARs) α, β and γ activate the transcription of multiple genes involved in lipid metabolism. Several natural and synthetic ligands have been identified for each PPAR isotype but little is known about the phosphorylation state of these receptors. We show here that activators of protein kinase A (PKA) can enhance mouse PPAR activity in the absence and the presence of exogenous ligands in transient transfection experiments. The activation function 1 (AF-1) of PPARs was dispensable for transcriptional enhancement, whereas the activation function 2 (AF-2) was required for this effect. We also show that several domains of PPAR can be phosphorylated by PKA in vitro. Moreover, gel experiments suggest that PKA stabilizes binding of the liganded PPAR to DNA. PKA inhibitors decreased not only the kinase dependent induction of PPARs but also their ligand-dependent induction, suggesting that the ligands may also mobilize the PKA pathway to lead to maximal transcriptional induction by PPARs. Moreover, comparing PPARα KO with PPARα wild-type mice, we show that the expression of the ACO gene can be regulated by PKA-activated PPARα in liver. These data demonstrate that the PKA pathway is an important modulator of PPAR activity and we propose a model associating this pathway in the control of fatty acid β-oxidation under conditions of fasting, stress and exercise. PMID:11117527

Vitamins and aging: pathways to NAD+ synthesis.

PubMed

Denu, John M

2007-05-04

Recent genetic evidence reveals additional salvage pathways for NAD(+) synthesis. In this issue, Belenky et al. (2007) report that nicotinamide riboside, a new NAD(+) precursor, regulates Sir2 deacetylase activity and life span in yeast. The ability of nicotinamide riboside to enhance life span does not depend on calorie restriction.

Oxygen-dependent regulation of c-di-GMP synthesis by SadC controls alginate production in Pseudomonas aeruginosa.

PubMed

Schmidt, Annika; Hammerbacher, Anna Silke; Bastian, Mike; Nieken, Karen Jule; Klockgether, Jens; Merighi, Massimo; Lapouge, Karine; Poschgan, Claudia; Kölle, Julia; Acharya, K Ravi; Ulrich, Martina; Tümmler, Burkhard; Unden, Gottfried; Kaever, Volkhard; Lory, Stephen; Haas, Dieter; Schwarz, Sandra; Döring, Gerd

2016-10-01

Pseudomonas aeruginosa produces increased levels of alginate in response to oxygen-deprived conditions. The regulatory pathway(s) that links oxygen limitation to increased synthesis of alginate has remained elusive. In the present study, using immunofluorescence microscopy, we show that anaerobiosis-induced alginate production by planktonic PAO1 requires the diguanylate cyclase (DGC) SadC, previously identified as a regulator of surface-associated lifestyles. Furthermore, we found that the gene products of PA4330 and PA4331, located in a predicted operon with sadC, have a major impact on alginate production: deletion of PA4330 (odaA, for oxygen-dependent alginate synthesis activator) caused an alginate production defect under anaerobic conditions, whereas a PA4331 (odaI, for oxygen-dependent alginate synthesis inhibitor) deletion mutant produced alginate also in the presence of oxygen, which would normally inhibit alginate synthesis. Based on their sequence, OdaA and OdaI have predicted hydratase and dioxygenase reductase activities, respectively. Enzymatic assays using purified protein showed that unlike OdaA, which did not significantly affect DGC activity of SadC, OdaI inhibited c-di-GMP production by SadC. Our data indicate that SadC, OdaA and OdaI are components of a novel response pathway of P. aeruginosa that regulates alginate synthesis in an oxygen-dependent manner. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

Pathogen Specific, IRF3-Dependent Signaling and Innate Resistance to Human Kidney Infection

PubMed Central

Fischer, Hans; Lutay, Nataliya; Ragnarsdóttir, Bryndís; Yadav, Manisha; Jönsson, Klas; Urbano, Alexander; Al Hadad, Ahmed; Rämisch, Sebastian; Storm, Petter; Dobrindt, Ulrich; Salvador, Ellaine; Karpman, Diana; Jodal, Ulf; Svanborg, Catharina

2010-01-01

The mucosal immune system identifies and fights invading pathogens, while allowing non-pathogenic organisms to persist. Mechanisms of pathogen/non-pathogen discrimination are poorly understood, as is the contribution of human genetic variation in disease susceptibility. We describe here a new, IRF3-dependent signaling pathway that is critical for distinguishing pathogens from normal flora at the mucosal barrier. Following uropathogenic E. coli infection, Irf3−/− mice showed a pathogen-specific increase in acute mortality, bacterial burden, abscess formation and renal damage compared to wild type mice. TLR4 signaling was initiated after ceramide release from glycosphingolipid receptors, through TRAM, CREB, Fos and Jun phosphorylation and p38 MAPK-dependent mechanisms, resulting in nuclear translocation of IRF3 and activation of IRF3/IFNβ-dependent antibacterial effector mechanisms. This TLR4/IRF3 pathway of pathogen discrimination was activated by ceramide and by P-fimbriated E. coli, which use ceramide-anchored glycosphingolipid receptors. Relevance of this pathway for human disease was supported by polymorphic IRF3 promoter sequences, differing between children with severe, symptomatic kidney infection and children who were asymptomatic bacterial carriers. IRF3 promoter activity was reduced by the disease-associated genotype, consistent with the pathology in Irf3−/− mice. Host susceptibility to common infections like UTI may thus be strongly influenced by single gene modifications affecting the innate immune response. PMID:20886096

SKF83959 Produces Antidepressant Effects in a Chronic Social Defeat Stress Model of Depression through BDNF-TrkB Pathway

PubMed Central

Jiang, Bo; Wang, Fang; Yang, Si; Fang, Peng; Deng, Zhi-Fang; Xiao, Jun-Li; Hu, Zhuang-Li

2015-01-01

Background: SKF83959 stimulates the phospholipase Cβ/inositol phosphate 3 pathway, resulting in the activation of Ca2+/calmodulin-dependent kinase IIα, which affects the synthesis of brain-derived neurotrophic factor, a neurotrophic factor critical for the pathophysiology of depression. Previous reports showed that SKF83959 elicited antidepressant activity in the forced swim test and tail suspension test as a novel triple reuptake inhibitor. However, there are no studies showing the effects of SKF83959 in a chronic stress model of depression and the role of phospholipase C/inositol phosphate 3/calmodulin-dependent kinase IIα/brain-derived neurotrophic factor pathway in SKF83959-mediated antidepressant effects. Methods: In this study, SKF83959 was firstly investigated in the chronic social defeat stress model of depression. The changes in hippocampal neurogenesis, dendrite spine density, and brain-derived neurotrophic factor signaling pathway after chronic social defeat stress and SKF83959 treatment were then investigated. Pharmacological inhibitors and small interfering RNA/short hairpin RNA methods were further used to explore the antidepressive mechanisms of SKF83959. Results: We found that SKF83959 produced antidepressant effects in the chronic social defeat stress model and also restored the chronic social defeat stress-induced decrease in hippocampal brain-derived neurotrophic factor signaling pathway, dendritic spine density, and neurogenesis. By using various inhibitors and siRNA/shRNA methods, we further demonstrated that the hippocampal dopamine D5 receptor, phospholipase C/inositol phosphate 3/ calmodulin-dependent kinase IIα pathway, and brain-derived neurotrophic factor system are all necessary for the SKF83959 effects. Conclusion: These results suggest that SKF83959 can be developed as a novel antidepressant and produces antidepressant effects via the hippocampal D5/ phospholipase C/inositol phosphate 3/calmodulin-dependent kinase IIα/brain-derived neurotrophic factor pathway. PMID:25522427

The cell cycle and acute kidney injury

PubMed Central

Price, Peter M.; Safirstein, Robert L.; Megyesi, Judit

2009-01-01

Acute kidney injury (AKI) activates pathways of cell death and cell proliferation. Although seemingly discrete and unrelated mechanisms, these pathways can now be shown to be connected and even to be controlled by similar pathways. The dependence of the severity of renal-cell injury on cell cycle pathways can be used to control and perhaps to prevent acute kidney injury. This review is written to address the correlation between cellular life and death in kidney tubules, especially in acute kidney injury. PMID:19536080

Mirtazapine exerts an anxiolytic-like effect through activation of the median raphe nucleus-dorsal hippocampal 5-HT pathway in contextual fear conditioning in rats.

PubMed

An, Yan; Chen, Chong; Inoue, Takeshi; Nakagawa, Shin; Kitaichi, Yuji; Wang, Ce; Izumi, Takeshi; Kusumi, Ichiro

2016-10-03

The functional role of serotonergic projections from the median raphe nucleus (MRN) to the dorsal hippocampus (DH) in anxiety remains understood poorly. The purpose of the present research was to examine the functional role of this pathway, using the contextual fear conditioning (CFC) model of anxiety. We show that intra-MRN microinjection of mirtazapine, a noradrenergic and specific serotonergic antidepressant, reduced freezing in CFC without affecting general motor activity dose-dependently, suggesting an anxiolytic-like effect. In addition, intra-MRN microinjection of mirtazapine dose-dependently increased extracellular concentrations of serotonin (5-HT) but not dopamine in the DH. Importantly, intra-DH pre-microinjection of WAY-100635, a 5-HT1A antagonist, significantly attenuated the effect of mirtazapine on freezing. These results, for the first time, suggest that activation of the MRN-DH 5-HT1A pathway exerts an anxiolytic-like effect in CFC. This is consistent with the literature that the hippocampus is essential for retrieval of contextual memory and that 5-HT1A receptor activation in the hippocampus primarily exerts an inhibitory effect on the neuronal activity. Copyright © 2016 Elsevier Inc. All rights reserved.

In Vitro Estrogenic and Breast Cancer Inhibitory Activities of Chemical Constituents Isolated from Rheum undulatum L.

PubMed

Lee, Dahae; Park, SeonJu; Choi, Sungyoul; Kim, Seung Hyun; Kang, Ki Sung

2018-05-18

We investigated the estrogenic and breast cancer inhibitory activities of chemical constituents isolated from Rhei undulati Rhizoma (roots of Rheum undulatum L.), which is used as a laxative, an anti-inflammatory, and an anti-blood stagnation agent. Estrogen-like activity was studied using the well characterized E-screen assay in estrogen receptor (ER)-positive MCF-7 cells. The mechanism underlying the breast cancer inhibitory activity of the compounds was studied using human ER-negative MDA-MB-231 and ER-positive MCF-7 cells. The activation of apoptosis pathway-related proteins was investigated by western blotting, using extracts of R. undulatum prepared in three solvent conditions (EX1, EX2, and EX3). The R. undulatum chemical constituents (compounds 1 ⁻ 3 ) showed estrogen-like activity in the concentration range of 10 to 50 μM, by increasing the proliferation of human ER-positive MCF-7 cells. These effects were attenuated by co-treatment with 100 nM fulvestrant, an ER antagonist. Compounds 1 ⁻ 3 decreased the viability of MCF-7 cells in a concentration-dependent manner. Compounds 1 (aloe emodin) and 2 (rhapontigenin) induced mitochondria-independent apoptosis by activating the caspase-8 pathway, whereas the cytotoxic effect of compound 3 (chrysophanol 1- O -β-d-glucopyranoside) was mediated through the mitochondria-dependent apoptotic pathway.

Epstein-Barr virus associated modulation of Wnt pathway is not dependent on latent membrane protein-1.

PubMed

Webb, Natasha; Connolly, Geoff; Tellam, Judy; Yap, Alpha S; Khanna, Rajiv

2008-09-22

Previous studies have indicated that Epstein-Barr virus (EBV) can modulate the Wnt pathway in virus-infected cells and this effect is mediated by EBV-encoded oncogene latent membrane protein 1 (LMP1). Here we have reassessed the role of LMP1 in regulating the expression of various mediators of the canonical Wnt cascade. Contradicting the previous finding, we found that the levels of E-cadherin, beta-catenin, Glycogen Synthase Kinase 3ss (GSK3beta), axin and alpha-catenin were not affected by the expression of LMP1 sequences from normal B cells or nasopharyngeal carcinoma. Moreover, we also show that LMP1 expression had no detectable effect on the E-cadherin and beta-catenin interaction and did not induce transcriptional activation of beta-catenin. Taken together these studies demonstrate that EBV-mediated activation of Wnt pathway is not dependent on the expression of LMP1.

Lactobacillus gasseri in the Upper Small Intestine Impacts an ACSL3-Dependent Fatty Acid-Sensing Pathway Regulating Whole-Body Glucose Homeostasis.

PubMed

Bauer, Paige V; Duca, Frank A; Waise, T M Zaved; Dranse, Helen J; Rasmussen, Brittany A; Puri, Akshita; Rasti, Mozhgan; O'Brien, Catherine A; Lam, Tony K T

2018-03-06

Long-chain acyl-CoA synthetase (ACSL)-dependent upper small intestinal lipid metabolism activates pre-absorptive pathways to regulate metabolic homeostasis, but whether changes in the upper small intestinal microbiota alter specific fatty acid-dependent pathways to impact glucose homeostasis remains unknown. We here first find that upper small intestinal infusion of Intralipid, oleic acid, or linoleic acid pre-absorptively increases glucose tolerance and lowers glucose production in rodents. High-fat feeding impairs pre-absorptive fatty acid sensing and reduces upper small intestinal Lactobacillus gasseri levels and ACSL3 expression. Transplantation of healthy upper small intestinal microbiota to high-fat-fed rodents restores L. gasseri levels and fatty acid sensing via increased ACSL3 expression, while L. gasseri probiotic administration to non-transplanted high-fat-fed rodents is sufficient to restore upper small intestinal ACSL3 expression and fatty acid sensing. In summary, we unveil a glucoregulatory role of upper small intestinal L. gasseri that impacts an ACSL3-dependent glucoregulatory fatty acid-sensing pathway. Copyright © 2018 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mu, Shumin; Hospital Affiliated to Shandong Traditional Chinese Medicine University, Jinan 250011; Institute of Endocrinology, Shandong Academy of Clinical Medicine, Jinan 250021

Highlights: Black-Right-Pointing-Pointer Diosgenin induces apoptosis in IGF-1-treated thyrocytes through two caspase pathways. Black-Right-Pointing-Pointer Diosgenin inhibits FLIP and activates caspase-8 in FAS related-pathway. Black-Right-Pointing-Pointer Diosgenin increases ROS, regulates the ratio of Bax/Bcl-2 in mitochondrial pathway. -- Abstract: Insulin-like growth factor-1 (IGF-1) is a growth factor of the thyroid that has been shown in our previous study to possess proliferative and antiapoptotic effects in FRTL-5 cell lines through the upregulation of cyclin D and Fas-associated death domain-like interleukin-1-converting enzyme (FLICE)-inhibitory protein (FLIP). Diosgenin, a natural steroid sapogenin from plants, has been shown to induce apoptosis in many cell lines, with the exceptionmore » of thyroid cells. In this report, we investigated the apoptotic effect and mechanism of diosgenin in IGF-1-stimulated primary human thyrocytes. Primary human thyrocytes were preincubated with or without IGF-1 for 24 h and subsequently exposed to varying concentrations of diosgenin for different times. We found that diosgenin induced apoptosis in human thyrocytes pretreated with IGF-1 in a dose-dependent manner through the activation of caspase cascades. Moreover, diosgenin inhibited FLIP and activated caspase-8 in the FAS-related apoptotic pathway. Diosgenin increased the production of ROS, regulated the balance of Bax and Bcl-2 and cleaved caspase-9 in the mitochondrial apoptotic pathway. These results indicate that diosgenin induces apoptosis in IGF-1-stimulated primary human thyrocytes through two caspase-dependent pathways.« less

Parathyroid Hormone Activates Phospholipase C (PLC)-Independent Protein Kinase C Signaling Pathway via Protein Kinase A (PKA)-Dependent Mechanism: A New Defined Signaling Route Would Induce Alternative Consideration to Previous Conceptions

PubMed Central

Tong, Guojun; Meng, Yue; Hao, Song; Hu, Shaoyu; He, Youhua; Yan, Wenjuan; Yang, Dehong

2017-01-01

Background Parathyroid hormone (PTH) is an effective anti-osteoporosis agent, after binding to its receptor PTHR1, several signaling pathways, including cAMP/protein kinase A (PKA) and phospholipase C (PLC)/protein kinase C (PKC), are initiated through G proteins; with the cAMP/PKA pathway as the major pathway. Earlier studies have reported that PTHR1 might also activate PKC via a PLC-independent mechanism, but this pathway remains unclear. Material/Methods In HEK293 cells, cAMP accumulation was measured with ELISA and PKC was measured with fluorescence resonance energy transfer (FRET) analysis using CKAR plasmid. In MC3T3-E1 cells, real-time PCR was performed to examine gene expressions. Then assays for cell apoptosis, cell differentiation, alkaline phosphatase activity, and mineralization were performed. Results The FRET analysis found that PTH(1–34), [G1,R19]PTH(1–34) (GR(1–34), and [G1,R19]PTH(1–28) (GR(1–28) were all activated by PKC. The PKC activation ability of GR(1–28) was blocked by cAMP inhibitor (Rp-cAMP) and rescued with the addition of active PKA-α and PKA-β. The PKC activation ability of GR(1–34) was partially inhibited by Rp-cAMP. In MC3T3-E1 cells, gene expressions of ALP, CITED1, NR4a2, and OSX that was regulated by GR(1–28) were significantly changed by the pan-PKC inhibitor Go6983. After pretreatment with Rp-cAMP, the gene expressions of ALP, CITED1, and OPG were differentially regulated by GR(1–28) or GR(1–34), and the difference was blunted by Go6983. PTH(1–34), GR(1–28), and GR(1–34) significantly decreased early apoptosis and augmented osteoblastic differentiation in accordance with the activities of PKA and PKC. Conclusions PLC-independent PKC activation induced by PTH could be divided into two potential mechanisms: one was PKA-dependent and associated with PTH(1–28); the other was PKA-independent and associated with PTH(29–34). We also found that PTH could activate PLC-independent PKC via PKA-dependent mechanisms. PMID:28424452

Phospholipase C/protein kinase C pathway mediates angiotensin II-dependent apoptosis in neonatal rat cardiac fibroblasts expressing AT1 receptor.

PubMed

Vivar, Raul; Soto, Cristian; Copaja, Miguel; Mateluna, Francisca; Aranguiz, Pablo; Muñoz, Juan Pablo; Chiong, Mario; Garcia, Lorena; Letelier, Alan; Thomas, Walter G; Lavandero, Sergio; Díaz-Araya, Guillermo

2008-08-01

Cardiac fibroblasts are the major non-myocyte cell constituent in the myocardium, and they are involved in heart remodeling. Angiotensin II type 1 receptor (AT1R) mediates the established actions of angiotensin II (Ang II), and changes in its expression have been reported in cardiac fibroblasts after myocardial infarction. However, the AT1R-dependent signaling pathways involved in cardiac fibroblast death remain unknown. Using adenovirus, we ectopically expressed AT1R in cultured neonatal rat cardiac fibroblasts and investigated the role of the phospholipase (PLC)/protein kinase C (PKC) pathway on Ang II-dependent death. Ang II induced cardiac fibroblast death characterized by an early loss of mitochondrial membrane potential, increased Bax/Bcl-2 ratio, caspase-3 activation, and DNA fragmentation. All these effects were prevented by the AT1R antagonist losartan, PLC inhibitor U73122, and PKC inhibitor Gö6976. We conclude that Ang II stimulates the intrinsic apoptotic pathway in cultured cardiac fibroblasts by the AT1R/PLC/PKC signaling pathway.

Protein C receptor stimulates multiple signaling pathways in breast cancer cells.

PubMed

Wang, Daisong; Liu, Chunye; Wang, Jingqiang; Jia, Yingying; Hu, Xin; Jiang, Hai; Shao, Zhi-Ming; Zeng, Yi Arial

2018-01-26

The protein C receptor (PROCR) has emerged as a stem cell marker in several normal tissues and has also been implicated in tumor progression. However, the functional role of PROCR and the signaling mechanisms downstream of PROCR remain poorly understood. Here, we dissected the PROCR signaling pathways in breast cancer cells. Combining protein array, knockdown, and overexpression methods, we found that PROCR concomitantly activates multiple pathways. We also noted that PROCR-dependent ERK and PI3k-Akt-mTOR signaling pathways proceed through Src kinase and transactivation of insulin-like growth factor 1 receptor (IGF-1R). These pathway activities led to the accumulation of c-Myc and cyclin D1. On the other hand, PROCR-dependent RhoA-ROCK-p38 signaling relied on coagulation factor II thrombin receptor (F2R). We confirmed these findings in primary cells isolated from triple-negative breast cancer-derived xenografts (PDX) that have high expression of PROCR. To the best our knowledge, this is the first comprehensive study of PROCR signaling in breast cancer cells, and its findings also shed light on the molecular mechanisms of PROCR in stem cells in normal tissue. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Airborne nitro-PAHs induce Nrf2/ARE defense system against oxidative stress and promote inflammatory process by activating PI3K/Akt pathway in A549 cells.

PubMed

Shang, Yu; Zhou, Qian; Wang, Tiantian; Jiang, Yuting; Zhong, Yufang; Qian, Guangren; Zhu, Tong; Qiu, Xinghua; An, Jing

2017-10-01

Ambient particulate matter (PM) is a worldwide health issue of concern. However, limited information is available regarding the toxic contributions of the nitro-derivatives of polycyclic aromatic hydrocarbons (nitro-PAHs). This study intend to examine whether 1-nitropyrene (1-NP) and 3-nitrofluoranthene (3-NF) could activate the nuclear factor-erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) antioxidant defense system, and whether the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway participates in regulating pro-inflammatory responses in A549 cells. Firstly, 1-NP and 3-NF concentration-dependently induced cellular apoptosis, reactive oxygen species (ROS) generation, DNA damage, S phase cell cycle arrest and differential expression of related cytokine genes. Secondly, 1-NP and 3-NF activated the Nrf2/ARE defense system, as evidenced by increased protein expression levels and nuclear translocation of transcription factor Nrf2, elevated Nrf2/ARE binding activity, up-regulated expression of the target gene heme oxygenase-1 (HO-1). Significantly increased protein expression of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and phosphorylation level of Akt indicated that the PI3K/Akt pathway was activated during pro-inflammatory process. Further, both PI3K inhibitor (LY294002) and Akt inhibitor (MK-2206) reversed the elevated TNF-α expression to control level. Our results suggested that Nrf2/ARE pathway activation might cause an initiation step in cellular protection against oxidative stress caused by nitro-PAHs, and the PI3K/Akt pathway participated in regulating inflammatory responses. Copyright © 2017 Elsevier Ltd. All rights reserved.

An ATM-independent S-phase checkpoint response involves CHK1 pathway

NASA Technical Reports Server (NTRS)

Zhou, Xiang-Yang; Wang, Xiang; Hu, Baocheng; Guan, Jun; Iliakis, George; Wang, Ya

2002-01-01

After exposure to genotoxic stress, proliferating cells actively slow down the DNA replication through a S-phase checkpoint to provide time for repair. We report that in addition to the ataxia-telangiectasia mutated (ATM)-dependent pathway that controls the fast response, there is an ATM-independent pathway that controls the slow response to regulate the S-phase checkpoint after ionizing radiation in mammalian cells. The slow response of S-phase checkpoint, which is resistant to wortmannin, sensitive to caffeine and UCN-01, and related to cyclin-dependent kinase phosphorylation, is much stronger in CHK1 overexpressed cells, and it could be abolished by Chk1 antisense oligonucleotides. These results provide evidence that the ATM-independent slow response of S-phase checkpoint involves CHK1 pathway.

ERK1/2 signalling protects against apoptosis following endoplasmic reticulum stress but cannot provide long-term protection against BAX/BAK-independent cell death.

PubMed

Darling, Nicola J; Balmanno, Kathryn; Cook, Simon J

2017-01-01

Disruption of protein folding in the endoplasmic reticulum (ER) causes ER stress. Activation of the unfolded protein response (UPR) acts to restore protein homeostasis or, if ER stress is severe or persistent, drive apoptosis, which is thought to proceed through the cell intrinsic, mitochondrial pathway. Indeed, cells that lack the key executioner proteins BAX and BAK are protected from ER stress-induced apoptosis. Here we show that chronic ER stress causes the progressive inhibition of the extracellular signal-regulated kinase (ERK1/2) signalling pathway. This is causally related to ER stress since reactivation of ERK1/2 can protect cells from ER stress-induced apoptosis whilst ERK1/2 pathway inhibition sensitises cells to ER stress. Furthermore, cancer cell lines harbouring constitutively active BRAFV600E are addicted to ERK1/2 signalling for protection against ER stress-induced cell death. ERK1/2 signalling normally represses the pro-death proteins BIM, BMF and PUMA and it has been proposed that ER stress induces BIM-dependent cell death. We found no evidence that ER stress increased the expression of these proteins; furthermore, BIM was not required for ER stress-induced death. Rather, ER stress caused the PERK-dependent inhibition of cap-dependent mRNA translation and the progressive loss of pro-survival proteins including BCL2, BCLXL and MCL1. Despite these observations, neither ERK1/2 activation nor loss of BAX/BAK could confer long-term clonogenic survival to cells exposed to ER stress. Thus, ER stress induces cell death by at least two biochemically and genetically distinct pathways: a classical BAX/BAK-dependent apoptotic response that can be inhibited by ERK1/2 signalling and an alternative ERK1/2- and BAX/BAK-independent cell death pathway.

Knockdown of hepatoma-derived growth factor-related protein-3 induces apoptosis of H1299 cells via ROS-dependent and p53-independent NF-κB activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yun, Hong Shik; Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 133-791; Baek, Jeong-Hwa

2014-07-11

Highlights: • HRP-3 is a radiation- and anticancer drug-responsive protein in H1299 cells. • Depletion of HRP-3 induces apoptosis of radio- and chemoresistant H1299 cells. • Depletion of HRP-3 promotes ROS generation via inhibition of the Nrf2/HO-1 pathway. • ROS generation enhances NF-κB activity, which acts as an upstream signal in the c-Myc/Noxa apoptotic pathway. - Abstract: We previously identified hepatoma-derived growth factor-related protein-3 (HRP-3) as a radioresistant biomarker in p53 wild-type A549 cells and found that p53-dependent induction of the PUMA pathway was a critical event in regulating the radioresistant phenotype. Here, we found that HRP-3 knockdown regulates themore » radioresistance of p53-null H1299 cells through a distinctly different molecular mechanism. HRP-3 depletion was sufficient to cause apoptosis of H1299 cells by generating substantial levels of reactive oxygen species (ROS) through inhibition of the Nrf2/HO-1 antioxidant pathway. Subsequent, ROS-dependent and p53-independent NF-κB activation stimulated expression of c-Myc and Noxa proteins, thereby inducing the apoptotic machinery. Our results thus extend the range of targets for the development of new drugs to treat both p53 wild-type or p53-null radioresistant lung cancer cells.« less

WASH and Tsg101/ALIX-dependent diversion of stress-internalized EGFR from the canonical endocytic pathway

PubMed Central

Tomas, Alejandra; Vaughan, Simon O.; Burgoyne, Thomas; Sorkin, Alexander; Hartley, John A.; Hochhauser, Daniel; Futter, Clare E.

2015-01-01

Stress exposure triggers ligand-independent EGF receptor (EGFR) endocytosis, but its post-endocytic fate and role in regulating signalling are unclear. We show that the p38 MAP kinase-dependent, EGFR tyrosine kinase (TK)-independent EGFR internalization induced by ultraviolet light C (UVC) or the cancer therapeutic cisplatin, is followed by diversion from the canonical endocytic pathway. Instead of lysosomal degradation or plasma membrane recycling, EGFR accumulates in a subset of LBPA-rich perinuclear multivesicular bodies (MVBs) distinct from those carrying EGF-stimulated EGFR. Stress-internalized EGFR co-segregates with exogenously expressed pre-melanosomal markers OA1 and fibrillar PMEL, following early endosomal sorting by the actin polymerization-promoting WASH complex. Stress-internalized EGFR is retained intracellularly by continued p38 activity in a mechanism involving ubiquitin-independent, ESCRT/ALIX-dependent incorporation onto intraluminal vesicles (ILVs) of MVBs. In contrast to the internalization-independent EGF-stimulated activation, UVC/cisplatin-triggered EGFR activation depends on EGFR internalization and intracellular retention. EGFR signalling from this MVB subpopulation delays apoptosis and might contribute to chemoresistance. PMID:26066081

Transcriptional and translational control of ornithine decarboxylase during Ras transformation.

PubMed Central

Shantz, Lisa M

2004-01-01

ODC (ornithine decarboxylase) activity is induced following ras activation. However, the Ras effector pathways responsible are unknown. These experiments used NIH-3T3 cells expressing partial-loss-of-function Ras mutants to activate selectively pathways downstream of Ras and examined the contribution of each pathway to ODC induction. Overexpression of Ras12V, a constitutively active mutant, resulted in ODC activities up to 20-fold higher than controls. Stable transfections of Ras partial-loss-of-function mutants and constitutively active forms of MEK (MAPK kinase) and Akt indicated that activation of more than one Ras effector pathway is necessary for the complete induction of ODC activity. The increase in ODC activity in Ras12V-transformed cells is not owing to a substantial change in ODC protein half-life, which increased by <2-fold. Northern-blot analysis and reporter assays suggested that the mechanism of ODC induction involves both a modest increase in the transcription of ODC mRNA and a much more considerable increase in the translation of mRNA into protein. ODC transcription was controlled through a pathway dependent on Raf/MEK/ERK (where ERK stands for extracellular-signal-regulated kinase) activation, whereas activation of the phosphoinositide 3-kinase and the Raf/MEK/ERK pathways were necessary for translational regulation of ODC. The increase in ODC synthesis was accompanied by changes in phosphorylation of eukaryotic initiation factor 4E and its binding protein 4E-BP1. Results show that the phosphoinositide 3-kinase pathway regulates phosphorylation of both proteins, whereas the Raf/MEK/ERK pathway affects only the eukaryotic initiation factor 4E phosphorylation. PMID:14519103

Phenformin activates the unfolded protein response in an AMP-activated protein kinase (AMPK)-dependent manner.

PubMed

Yang, Liu; Sha, Haibo; Davisson, Robin L; Qi, Ling

2013-05-10

The cross-talk between UPR activation and metabolic stress remains largely unclear. Phenformin treatment activates the IRE1α and PERK pathways in an AMPK-dependent manner. AMPK is required for phenformin-mediated IRE1α and PERK activation. Our findings demonstrate the cross-talk between UPR and metabolic signals. Activation of the unfolded protein response (UPR) is associated with the disruption of endoplasmic reticulum (ER) homeostasis and has been implicated in the pathogenesis of many human metabolic diseases, including obesity and type 2 diabetes. However, the nature of the signals activating UPR under these conditions remains largely unknown. Using a method that we recently optimized to directly measure UPR sensor activation, we screened the effect of various metabolic drugs on UPR activation and show that the anti-diabetic drug phenformin activates UPR sensors IRE1α and pancreatic endoplasmic reticulum kinase (PERK) in both an ER-dependent and ER-independent manner. Mechanistically, AMP-activated protein kinase (AMPK) activation is required but not sufficient to initiate phenformin-mediated IRE1α and PERK activation, suggesting the involvement of additional factor(s). Interestingly, activation of the IRE1α (but not PERK) pathway is partially responsible for the cytotoxic effect of phenformin. Together, our data show the existence of a non-canonical UPR whose activation requires the cytosolic kinase AMPK, adding another layer of complexity to UPR activation upon metabolic stress.

The functional interplay between the HIF pathway and the ubiquitin system - more than a one-way road.

PubMed

Günter, Julia; Ruiz-Serrano, Amalia; Pickel, Christina; Wenger, Roland H; Scholz, Carsten C

2017-07-15

The hypoxia inducible factor (HIF) pathway and the ubiquitin system represent major cellular processes that are involved in the regulation of a plethora of cellular signaling pathways and tissue functions. The ubiquitin system controls the ubiquitination of proteins, which is the covalent linkage of one or several ubiquitin molecules to specific targets. This ubiquitination is catalyzed by approximately 1000 different E3 ubiquitin ligases and can lead to different effects, depending on the type of internal ubiquitin chain linkage. The best-studied function is the targeting of proteins for proteasomal degradation. The activity of E3 ligases is antagonized by proteins called deubiquitinases (or deubiquitinating enzymes), which negatively regulate ubiquitin chains. This is performed in most cases by the catalytic removal of these chains from the targeted protein. The HIF pathway is regulated in an oxygen-dependent manner by oxygen-sensing hydroxylases. Covalent modification of HIFα subunits leads to the recruitment of an E3 ligase complex via the von Hippel-Lindau (VHL) protein and the subsequent polyubiquitination and proteasomal degradation of HIFα subunits, demonstrating the regulation of the HIF pathway by the ubiquitin system. This unidirectional effect of an E3 ligase on the HIF pathway is the best-studied example for the interplay between these two important cellular processes. However, additional regulatory mechanisms of the HIF pathway through the ubiquitin system are emerging and, more recently, also the reciprocal regulation of the ubiquitin system through components of the HIF pathway. Understanding these mechanisms and their relevance for the activity of each other is of major importance for the comprehensive elucidation of the oxygen-dependent regulation of cellular processes. This review describes the current knowledge of the functional bidirectional interplay between the HIF pathway and the ubiquitin system on the protein level. Copyright © 2017 Elsevier Inc. All rights reserved.

Epistatic role of base excision repair and mismatch repair pathways in mediating cisplatin cytotoxicity

PubMed Central

Kothandapani, Anbarasi; Sawant, Akshada; Dangeti, Venkata Srinivas Mohan Nimai; Sobol, Robert W.; Patrick, Steve M.

2013-01-01

Base excision repair (BER) and mismatch repair (MMR) pathways play an important role in modulating cis-Diamminedichloroplatinum (II) (cisplatin) cytotoxicity. In this article, we identified a novel mechanistic role of both BER and MMR pathways in mediating cellular responses to cisplatin treatment. Cells defective in BER or MMR display a cisplatin-resistant phenotype. Targeting both BER and MMR pathways resulted in no additional resistance to cisplatin, suggesting that BER and MMR play epistatic roles in mediating cisplatin cytotoxicity. Using a DNA Polymerase β (Polβ) variant deficient in polymerase activity (D256A), we demonstrate that MMR acts downstream of BER and is dependent on the polymerase activity of Polβ in mediating cisplatin cytotoxicity. MSH2 preferentially binds a cisplatin interstrand cross-link (ICL) DNA substrate containing a mismatch compared with a cisplatin ICL substrate without a mismatch, suggesting a novel mutagenic role of Polβ in activating MMR in response to cisplatin. Collectively, these results provide the first mechanistic model for BER and MMR functioning within the same pathway to mediate cisplatin sensitivity via non-productive ICL processing. In this model, MMR participation in non-productive cisplatin ICL processing is downstream of BER processing and dependent on Polβ misincorporation at cisplatin ICL sites, which results in persistent cisplatin ICLs and sensitivity to cisplatin. PMID:23761438

Metabolomic studies identify changes in transmethylation and polyamine metabolism in a brain-specific mouse model of tuberous sclerosis complex.

PubMed

McKenna, James; Kapfhamer, David; Kinchen, Jason M; Wasek, Brandi; Dunworth, Matthew; Murray-Stewart, Tracy; Bottiglieri, Teodoro; Casero, Robert A; Gambello, Michael J

2018-06-15

Tuberous sclerosis complex (TSC) is an autosomal dominant neurodevelopmental disorder and the quintessential disorder of mechanistic Target of Rapamycin Complex 1 (mTORC1) dysregulation. Loss of either causative gene, TSC1 or TSC2, leads to constitutive mTORC1 kinase activation and a pathologically anabolic state of macromolecular biosynthesis. Little is known about the organ-specific metabolic reprogramming that occurs in TSC-affected organs. Using a mouse model of TSC in which Tsc2 is disrupted in radial glial precursors and their neuronal and glial descendants, we performed an unbiased metabolomic analysis of hippocampi to identify Tsc2-dependent metabolic changes. Significant metabolic reprogramming was found in well-established pathways associated with mTORC1 activation, including redox homeostasis, glutamine/tricarboxylic acid cycle, pentose and nucleotide metabolism. Changes in two novel pathways were identified: transmethylation and polyamine metabolism. Changes in transmethylation included reduced methionine, cystathionine, S-adenosylmethionine (SAM-the major methyl donor), reduced SAM/S-adenosylhomocysteine ratio (cellular methylation potential), and elevated betaine, an alternative methyl donor. These changes were associated with alterations in SAM-dependent methylation pathways and expression of the enzymes methionine adenosyltransferase 2A and cystathionine beta synthase. We also found increased levels of the polyamine putrescine due to increased activity of ornithine decarboxylase, the rate-determining enzyme in polyamine synthesis. Treatment of Tsc2+/- mice with the ornithine decarboxylase inhibitor α-difluoromethylornithine, to reduce putrescine synthesis dose-dependently reduced hippocampal astrogliosis. These data establish roles for SAM-dependent methylation reactions and polyamine metabolism in TSC neuropathology. Importantly, both pathways are amenable to nutritional or pharmacologic therapy.

A rapid form of activity-dependent recovery from short-term synaptic depression in the intensity pathway of the auditory brainstem

PubMed Central

Horiuchi, Timothy K.

2011-01-01

Short-term synaptic plasticity acts as a time- and firing rate-dependent filter that mediates the transmission of information across synapses. In the avian auditory brainstem, specific forms of plasticity are expressed at different terminals of the same auditory nerve fibers and contribute to the divergence of acoustic timing and intensity information. To identify key differences in the plasticity properties, we made patch-clamp recordings from neurons in the cochlear nucleus responsible for intensity coding, nucleus angularis, and measured the time course of the recovery of excitatory postsynaptic currents following short-term synaptic depression. These synaptic responses showed a very rapid recovery, following a bi-exponential time course with a fast time constant of ~40 ms and a dependence on the presynaptic activity levels, resulting in a crossing over of the recovery trajectories following high-rate versus low-rate stimulation trains. We also show that the recorded recovery in the intensity pathway differs from similar recordings in the timing pathway, specifically the cochlear nucleus magnocellularis, in two ways: (1) a fast recovery that was not due to recovery from postsynaptic receptor desensitization and (2) a recovery trajectory that was characterized by a non-monotonic bump that may be due in part to facilitation mechanisms more prevalent in the intensity pathway. We tested whether a previously proposed model of synaptic transmission based on vesicle depletion and sequential steps of vesicle replenishment could account for the recovery responses, and found it was insufficient, suggesting an activity-dependent feedback mechanism is present. We propose that the rapid recovery following depression allows improved coding of natural auditory signals that often consist of sound bursts separated by short gaps. PMID:21409439

Pulsed electromagnetic field inhibits RANKL-dependent osteoclastic differentiation in RAW264.7 cells through the Ca{sup 2+}-calcineurin-NFATc1 signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Jie; Xu, Haixia; Han, Zhongyu

Pulsed electromagnetic field (PEMF) has been reported to improve bone healing in osteoporosis patients. However, the precise mechanism has remained largely unknown. This study aimed to investigate the effects of PEMF on nuclear factor κB ligand (RANKL)-dependent osteoclastic differentiation and the Ca{sup 2+}-calcineurin-NFATc1 signaling pathway in RAW264.7 cells in vitro. Treating RAW264.7 cells with RANKL for 4 days induced osteoclastic differentiation in vitro, and the formation of multinucleated osteoclasts, bone resorption-pit formation, tartrate-resistant acid phosphatase (TRAP) activity and the protein levels of cathepsin K, TRAP, Nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) and matrix metalloproteinase 9 (MMP-9) were significantly decreased. The mRNA levelsmore » of specific genes related to osteoclastogenesis (TRAP, NFATc1, CTSK and MMP-9) were also reduced. Moreover, the oscillations of intracellular Ca{sup 2+} in RANKL-dependent RAW264.7 cells were suppressed by PEMF, as well as by inhibitors of membrane and store-operated Ca{sup 2+} channels. Meanwhile, calcineurin activity was increased, although its protein level was not changed. PEMF increased phospho-NFATc1 in the cytosol while suppressing the nuclear translocation of NFATc1, thus inhibiting osteoclastic differentiation by suppressing the Ca{sup 2+}-calcineurin-NFATc1 signaling pathway. Although many questions remain unresolved, to our knowledge, this is the first report demonstrating that PEMF is beneficial against RANKL-dependent osteoclastic differentiation in RAW264.7 cells in vitro via inhibiting the Ca{sup 2+}-calcineurin-NFATc1 signaling pathway.« less

Pulsed electromagnetic field inhibits RANKL-dependent osteoclastic differentiation in RAW264.7 cells through the Ca2+-calcineurin-NFATc1 signaling pathway.

PubMed

Zhang, Jie; Xu, Haixia; Han, Zhongyu; Chen, Ping; Yu, Qiang; Lei, Yutian; Li, Zongze; Zhao, Ming; Tian, Jing

2017-01-08

Pulsed electromagnetic field (PEMF) has been reported to improve bone healing in osteoporosis patients. However, the precise mechanism has remained largely unknown. This study aimed to investigate the effects of PEMF on nuclear factor κB ligand (RANKL)-dependent osteoclastic differentiation and the Ca 2+ -calcineurin-NFATc1 signaling pathway in RAW264.7 cells in vitro. Treating RAW264.7 cells with RANKL for 4 days induced osteoclastic differentiation in vitro, and the formation of multinucleated osteoclasts, bone resorption-pit formation, tartrate-resistant acid phosphatase (TRAP) activity and the protein levels of cathepsin K, TRAP, Nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) and matrix metalloproteinase 9 (MMP-9) were significantly decreased. The mRNA levels of specific genes related to osteoclastogenesis (TRAP, NFATc1, CTSK and MMP-9) were also reduced. Moreover, the oscillations of intracellular Ca 2+ in RANKL-dependent RAW264.7 cells were suppressed by PEMF, as well as by inhibitors of membrane and store-operated Ca 2+ channels. Meanwhile, calcineurin activity was increased, although its protein level was not changed. PEMF increased phospho-NFATc1 in the cytosol while suppressing the nuclear translocation of NFATc1, thus inhibiting osteoclastic differentiation by suppressing the Ca 2+ -calcineurin-NFATc1 signaling pathway. Although many questions remain unresolved, to our knowledge, this is the first report demonstrating that PEMF is beneficial against RANKL-dependent osteoclastic differentiation in RAW264.7 cells in vitro via inhibiting the Ca 2+ -calcineurin-NFATc1 signaling pathway. Copyright © 2016 Elsevier Inc. All rights reserved.

Detergent sclerosants at sub-lytic concentrations induce endothelial cell apoptosis through a caspase dependent pathway.

PubMed

Cooley-Andrade, Osvaldo; Cheung, Kelvin; Chew, An-Ning; Connor, David Ewan; Parsi, Kurosh

2016-07-01

To investigate the apoptotic effects of detergent sclerosants sodium tetradecylsulphate (STS) and polidocanol (POL) on endothelial cells at sub-lytic concentrations. Human umbilical vein endothelial cells (HUVECs) were isolated and labelled with antibodies to assess for apoptosis and examined with confocal microscopy and flow cytometry. Isolated HUVECs viability was assessed using propidium iodide staining. Early apoptosis was determined by increased phosphatidylserine exposure by lactadherin binding. Caspase 3, 8, 9 and Bax activation as well as inhibitory assays with Pan Caspase (Z-VAD-FMK) and Bax (BI-6C9) were assessed to identify apoptotic pathways. Porimin activation was used to assess cell membrane permeability. Cell lysis reached almost 100 % with STS at 0.3 % and with POL at 0.6 %. Apoptosis was seen with both STS and POL at concentrations ranging from 0.075 to 0.15 %. PS exposure increased with both STS and POL and exhibited a dose-dependent trend. Active Caspase 3, 8 and 9 but not Bax were increased in HUVECs stimulated with low concentrations of both STS and POL. Inhibitory assays demonstrated Caspase 3, 8, 9 inhibition at low concentrations (0.075 to 0.6 %) with both STS and POL. Both agents increased the activation of porimin at all concentrations. Both sclerosants induced endothelial cell (EC) apoptosis at sub-lytic concentrations through a caspase-dependant pathway. Both agents induced EC oncosis.

Activation of the yeast Hippo pathway by phosphorylation-dependent assembly of signaling complexes.

PubMed

Rock, Jeremy M; Lim, Daniel; Stach, Lasse; Ogrodowicz, Roksana W; Keck, Jamie M; Jones, Michele H; Wong, Catherine C L; Yates, John R; Winey, Mark; Smerdon, Stephen J; Yaffe, Michael B; Amon, Angelika

2013-05-17

Scaffold-assisted signaling cascades guide cellular decision-making. In budding yeast, one such signal transduction pathway called the mitotic exit network (MEN) governs the transition from mitosis to the G1 phase of the cell cycle. The MEN is conserved and in metazoans is known as the Hippo tumor-suppressor pathway. We found that signaling through the MEN kinase cascade was mediated by an unusual two-step process. The MEN kinase Cdc15 first phosphorylated the scaffold Nud1. This created a phospho-docking site on Nud1, to which the effector kinase complex Dbf2-Mob1 bound through a phosphoserine-threonine binding domain, in order to be activated by Cdc15. This mechanism of pathway activation has implications for signal transmission through other kinase cascades and might represent a general principle in scaffold-assisted signaling.

Pathway Activity Profiling (PAPi): from the metabolite profile to the metabolic pathway activity.

PubMed

Aggio, Raphael B M; Ruggiero, Katya; Villas-Bôas, Silas Granato

2010-12-01

Metabolomics is one of the most recent omics-technologies and uses robust analytical techniques to screen low molecular mass metabolites in biological samples. It has evolved very quickly during the last decade. However, metabolomics datasets are considered highly complex when used to relate metabolite levels to metabolic pathway activity. Despite recent developments in bioinformatics, which have improved the quality of metabolomics data, there is still no straightforward method capable of correlating metabolite level to the activity of different metabolic pathways operating within the cells. Thus, this kind of analysis still depends on extremely laborious and time-consuming processes. Here, we present a new algorithm Pathway Activity Profiling (PAPi) with which we are able to compare metabolic pathway activities from metabolite profiles. The applicability and potential of PAPi was demonstrated using a previously published data from the yeast Saccharomyces cerevisiae. PAPi was able to support the biological interpretations of the previously published observations and, in addition, generated new hypotheses in a straightforward manner. However, PAPi is time consuming to perform manually. Thus, we also present here a new R-software package (PAPi) which implements the PAPi algorithm and facilitates its usage to quickly compare metabolic pathways activities between different experimental conditions. Using the identified metabolites and their respective abundances as input, the PAPi package calculates pathways' Activity Scores, which represents the potential metabolic pathways activities and allows their comparison between conditions. PAPi also performs principal components analysis and analysis of variance or t-test to investigate differences in activity level between experimental conditions. In addition, PAPi generates comparative graphs highlighting up- and down-regulated pathway activity. These datasets are available in http://www.4shared.com/file/hTWyndYU/extra.html and http://www.4shared.com/file/VbQIIDeu/intra.html. PAPi package is available in: http://www.4shared.com/file/s0uIYWIg/PAPi_10.html s.villas-boas@auckland.ac.nz Supplementary data are available at Bioinformatics online.

2′,5′-Dihydroxychalcone-induced glutathione is mediated by oxidative stress and kinase signaling pathways

PubMed Central

Kachadourian, Remy; Pugazhenthi, Subbiah; Velmurugan, Kalpana; Backos, Donald S.; Franklin, Christopher C.; McCord, Joe M.; Day, Brian J.

2011-01-01

Hydroxychalcones are naturally occurring compounds that continue to attract considerable interest due to their anti-inflammatory and anti-angiogenic properties. They have been reported to inhibit the synthesis of the inducible nitric oxide (NO) synthase and to induce the expression of heme oxygenase-1 (HO-1). This study examines the mechanisms by which 2′,5′-dihydroxychalcone (2′,5′-DHC) induces an increase in cellular glutathione (GSH) levels using a cell line stably expressing a luciferase reporter gene driven by antioxidant response elements (MCF-7/AREc32). 2′,5′-DHC-induced increase in cellular GSH levels was partially inhibited by the catalytic antioxidant MnTDE-1,3-IP5+, suggesting that reactive oxygen species (ROS) mediate the antioxidant adaptive response. 2′,5′-DHC treatment induced the phosphorylation of c-Jun N-terminal kinase (JNK) pathway that was also inhibited by MnTDE-1,3-IP5+. These findings suggest a ROS-dependent activation of the AP-1 transcriptional response. However, while 2′,5′-DHC triggered the NF-E2-related factor 2 (Nrf2) transcriptional response, co-treatment with MnTDE-1,3-IP5+ did not decrease 2′,5′-DHC-induced Nrf2/ARE activity, showing that this pathway is not dependent on ROS. Moreover, pharmacological inhibitors of mitogen-activated protein (MAP) kinase pathways showed a role for JNK and p38MAPK in mediating the 2′,5′-DHC-induced Nrf2 response. These findings suggest that the 2′,5′-DHC-induced increase in GSH levels results from a combination of ROS-dependent and ROS-independent pathways. PMID:21712085

Iron-induced oxidative injury differentially regulates PI3K/Akt/GSK3beta pathway in synaptic endings from adult and aged rats.

PubMed

Uranga, Romina María; Giusto, Norma María; Salvador, Gabriela Alejandra

2009-10-01

In this work we study the state of phosphoinositide-3-kinase/Akt/glycogen synthase kinase 3 beta (PI3K/Akt/GSK3beta) signaling during oxidative injury triggered by free iron using cerebral cortex synaptic endings isolated from adult (4-month-old) and aged (28-month-old) rats. Synaptosomes were exposed to FeSO4 (50 microM) for different periods of time and synaptosomal viability and the state of the PI3K/Akt/GSK3beta pathway were evaluated in adult and aged animals. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction and lactate dehydrogenase leakage were significantly affected in both age groups. However, aged animals showed a greater susceptibility to oxidative stress. In adults, Akt was activated after a brief exposure time (5 min), whereas in aged animals activation occurred after 5 and 30 min of incubation with the metal ion. GSK3beta phosphorylation showed the same activation pattern as that observed for Akt. Both Akt and GSK3beta phosphorylation were dependent on PI3K activation. Extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation was temporally coincident with Akt activation and was PI3K dependent in adults, whereas ERK1/2 activation in aged rats was higher than that observed in adults and showed no dependence on PI3K activity. We demonstrate here that synaptic endings from adult and aged animals subjected to iron-induced neurotoxicity show a differential profile in the activation of PI3K/Akt/GSK3beta. Our results strongly suggest that the increased susceptibility of aged animals to oxidative injury provokes a differential modulation of key signaling pathways involved in synaptic plasticity and neuronal survival.

Activation of Cu,Zn-superoxide dismutase in the absence of oxygen and the copper chaperone CCS.

PubMed

Leitch, Jeffry M; Jensen, Laran T; Bouldin, Samantha D; Outten, Caryn E; Hart, P John; Culotta, Valeria C

2009-08-14

Eukaryotic Cu,Zn-superoxide dismutases (SOD1s) are generally thought to acquire the essential copper cofactor and intramolecular disulfide bond through the action of the CCS copper chaperone. However, several metazoan SOD1s have been shown to acquire activity in vivo in the absence of CCS, and the Cu,Zn-SOD from Caenorhabditis elegans has evolved complete independence from CCS. To investigate SOD1 activation in the absence of CCS, we compared and contrasted the CCS-independent activation of C. elegans and human SOD1 to the strict CCS-dependent activation of Saccharomyces cerevisiae SOD1. Using a yeast expression system, both pathways were seen to acquire copper derived from cell surface transporters and compete for the same intracellular pool of copper. Like CCS, CCS-independent activation occurs rapidly with a preexisting pool of apo-SOD1 without the need for new protein synthesis. The two pathways, however, strongly diverge when assayed for the SOD1 disulfide. SOD1 molecules that are activated without CCS exhibit disulfide oxidation in vivo without oxygen and under copper-depleted conditions. The strict requirement for copper, oxygen, and CCS in disulfide bond oxidation appears exclusive to yeast SOD1, and we find that a unique proline at position 144 in yeast SOD1 is responsible for this disulfide effect. CCS-dependent and -independent pathways also exhibit differential requirements for molecular oxygen. CCS activation of SOD1 requires oxygen, whereas the CCS-independent pathway is able to activate SOD1s even under anaerobic conditions. In this manner, Cu,Zn-SOD from metazoans may retain activity over a wide range of physiological oxygen tensions.

A Quantitative RNAi Screen for JNK Modifiers Identifies Pvr as a Novel Regulator of Drosophila Immune Signaling

PubMed Central

Bond, David; Foley, Edan

2009-01-01

Drosophila melanogaster responds to gram-negative bacterial challenges through the IMD pathway, a signal transduction cassette that is driven by the coordinated activities of JNK, NF-κB and caspase modules. While many modifiers of NF-κB activity were identified in cell culture and in vivo assays, the regulatory apparatus that determines JNK inputs into the IMD pathway is relatively unexplored. In this manuscript, we present the first quantitative screen of the entire genome of Drosophila for novel regulators of JNK activity in the IMD pathway. We identified a large number of gene products that negatively or positively impact on JNK activation in the IMD pathway. In particular, we identified the Pvr receptor tyrosine kinase as a potent inhibitor of JNK activation. In a series of in vivo and cell culture assays, we demonstrated that activation of the IMD pathway drives JNK-dependent expression of the Pvr ligands, Pvf2 and Pvf3, which in turn act through the Pvr/ERK MAP kinase pathway to attenuate the JNK and NF-κB arms of the IMD pathway. Our data illuminate a poorly understood arm of a critical and evolutionarily conserved innate immune response. Furthermore, given the pleiotropic involvement of JNK in eukaryotic cell biology, we believe that many of the novel regulators identified in this screen are of interest beyond immune signaling. PMID:19893628

Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR Cascade Inhibitors: How Mutations Can Result in Therapy Resistance and How to Overcome Resistance

PubMed Central

McCubrey, James A.; Steelman, Linda S.; Chappell, William H.; Abrams, Stephen L.; Franklin, Richard A.; Montalto, Giuseppe; Cervello, Melchiorre; Libra, Massimo; Candido, Saverio; Malaponte, Grazia; Mazzarino, Maria C.; Fagone, Paolo; Nicoletti, Ferdinando; Bäsecke, Jörg; Mijatovic, Sanja; Maksimovic-Ivanic, Danijela; Milella, Michele; Tafuri, Agostino; Chiarini, Francesca; Evangelisti, Camilla; Cocco, Lucio; Martelli, Alberto M.

2012-01-01

The Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR cascades are often activated by genetic alterations in upstream signaling molecules such as receptor tyrosine kinases (RTK). Targeting these pathways is often complex and can result in pathway activation depending on the presence of upstream mutations (e.g., Raf inhibitors induce Raf activation in cells with wild type (WT) RAF in the presence of mutant, activated RAS) and rapamycin can induce Akt activation. Targeting with inhibitors directed at two constituents of the same pathway or two different signaling pathways may be a more effective approach. This review will first evaluate potential uses of Raf, MEK, PI3K, Akt and mTOR inhibitors that have been investigated in pre-clinical and clinical investigations and then discuss how cancers can become insensitive to various inhibitors and potential strategies to overcome this resistance. PMID:23085539

Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR cascade inhibitors: how mutations can result in therapy resistance and how to overcome resistance.

PubMed

McCubrey, James A; Steelman, Linda S; Chappell, William H; Abrams, Stephen L; Franklin, Richard A; Montalto, Giuseppe; Cervello, Melchiorre; Libra, Massimo; Candido, Saverio; Malaponte, Grazia; Mazzarino, Maria C; Fagone, Paolo; Nicoletti, Ferdinando; Bäsecke, Jörg; Mijatovic, Sanja; Maksimovic-Ivanic, Danijela; Milella, Michele; Tafuri, Agostino; Chiarini, Francesca; Evangelisti, Camilla; Cocco, Lucio; Martelli, Alberto M

2012-10-01

The Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR cascades are often activated by genetic alterations in upstream signaling molecules such as receptor tyrosine kinases (RTK). Targeting these pathways is often complex and can result in pathway activation depending on the presence of upstream mutations (e.g., Raf inhibitors induce Raf activation in cells with wild type (WT) RAF in the presence of mutant, activated RAS) and rapamycin can induce Akt activation. Targeting with inhibitors directed at two constituents of the same pathway or two different signaling pathways may be a more effective approach. This review will first evaluate potential uses of Raf, MEK, PI3K, Akt and mTOR inhibitors that have been investigated in pre-clinical and clinical investigations and then discuss how cancers can become insensitive to various inhibitors and potential strategies to overcome this resistance.

A beta1-adrenergic receptor CaM kinase II-dependent pathway mediates cardiac myocyte fetal gene induction.

PubMed

Sucharov, Carmen C; Mariner, Peter D; Nunley, Karin R; Long, Carlin; Leinwand, Leslie; Bristow, Michael R

2006-09-01

Beta-adrenergic signaling plays an important role in the natural history of dilated cardiomyopathies. Chronic activation of beta-adrenergic receptors (beta1-AR and beta2-AR) during periods of cardiac stress ultimately harms the failing heart by mechanisms that include alterations in gene expression. Here, we show that stimulation of beta-ARs with isoproterenol in neonate rat ventricular myocytes causes a "fetal" response in the relative activities of the human cardiac fetal and/or adult gene promoters that includes repression of the human and rat alpha-myosin heavy chain (alpha-MyHC) promoters with simultaneous activation of the human atrial natriuretic peptide (ANP) and rat beta-MyHC promoters. We also show that the promoter changes correlate with changes in endogenous gene expression as measured by mRNA expression. Furthermore, we show that these changes are specifically mediated by the beta1-AR, but not the beta2-AR, and are independent of alpha1-AR stimulation. We also demonstrate that the fetal gene response is independent of cAMP and protein kinase A, whereas inhibition of Ca2+/calmodulin-dependent protein kinase (CaMK) pathway blocks isoproterenol-mediated fetal gene program induction. Finally, we show that induction of the fetal program is dependent on activation of the L-type Ca2+ channel. We conclude that in neonatal rat cardiac myocytes, agonist-occupied beta1-AR mobilizes Ca2+ stores to activate fetal gene induction through cAMP independent pathways that involve CaMK.

Activation of PI3K/AKT and MAPK Pathway through a PDGFRβ-Dependent Feedback Loop Is Involved in Rapamycin Resistance in Hepatocellular Carcinoma

PubMed Central

Yao, Li-Qing; Tan, Chang-Jun; Huang, Xiao-Yong; Ke, Ai-Wu; Dai, Zhi; Fan, Jia; Zhou, Jian

2012-01-01

Background Rapamycin is an attractive approach for the treatment and prevention of HCC recurrence after liver transplantation. However, the objective response rates of rapamycin achieved with single-agent therapy were modest, supporting that rapamycin resistance is a frequently observed characteristic of many cancers. Some studies have been devoted to understanding the mechanisms of rapamycin resistance, however, the mechanisms are cell-type-dependent and studies on rapamycin resistance in HCC are extremely limited. Methodology/Principal Findings The anti-tumor sensitivity of rapamycin was modest in vitro and in vivo. In both human and rat HCC cells, rapamycin up-regulated the expression and phosphorylation of PDGFRβ in a time and dose-dependent manner as assessed by RT-PCR and western blot analysis. Using siRNA mediated knockdown of PDGFRβ, we confirmed that subsequent activation of AKT and ERK was PDGFRβ-dependent and compromised the anti-tumor activity of rapamycin. Then, blockade of this PDGFRβ-dependent feedback loop by sorafenib enhanced the anti-tumor sensitivity of rapamycin in vitro and in an immunocompetent orthotopic rat model of HCC. Conclusions Activation of PI3K/AKT and MAPK pathway through a PDGFRβ-dependent feedback loop compromises the anti-tumor activity of rapamycin in HCC, and blockade of this feedback loop by sorafenib is an attractive approach to improve the anti-tumor effect of rapamycin, particularly in preventing or treating HCC recurrence after liver transplantation. PMID:22428038

A novel function for Wnt signaling modulating neuronal firing activity and the temporal structure of spontaneous oscillation in the entorhinal-hippocampal circuit.

PubMed

Oliva, Carolina A; Inestrosa, Nibaldo C

2015-07-01

During early and late postnatal developments, the establishment of functional neuronal connectivity depends on molecules like Wnt that help the recently formed synapses to establish and consolidate their new cellular interactions. However, unlike other molecules, whether Wnt can modulate the firing properties of cells is unknown. Here, for the first time we explore the physiological effect of the canonical and non-canonical Wnt pathways on a circuit that is currently generating oscillatory activity, the entorhinal cortex-hippocampal circuit. Our results indicate that Wnt pathways have strong influence in the circuital and cellular properties depending on the Wnt protein isoforms, concentration, and type of neuronal circuit. Antibodies against canonical and non-canonical ligands, as well as WASP-1 and sFRP-2, demonstrate that constitutive release of Wnts contributes to the maintenance of the network and intrinsic properties of the circuit. Furthermore, we found that the excess of Wnt3a or the permanent intracellular activation of the pathway with BIO-6 accelerates the period of the oscillation by disrupting the oscillatory units (Up states) in short units, presumably by affecting the synaptic mechanisms that couples neurons into the oscillatory cycle, but without affecting the spike generation. Instead, low doses of Wnt5a increase the period of the oscillation in EC by incorporating new cells into the network activity, probably modifying firing activity in other places of the circuit. Moreover, we found that Wnt signaling operates under different principles in the hippocampus. Using pyrvinium pamoate, a Wnt/β-catenin dependent pathway inhibitor, we demonstrated that this pathway is essential to keep the firing activity in the circuit CA3, and in less degree of CA1 circuit. However, CA1 circuit possesses homeostatic mechanisms to up-regulate the firing activity when it has been suppressed in CA3, and to down-modulate the cellular excitability when exacerbated circuital activity has dominated. In summary, the amount of Wnt that is being released can exert a fine tuning of the physiological output, modulating firing activity, improving reliability of communication between neurons, and maintaining a continuous self-regulatory cycle of synaptic structure-function that can be present during all postnatal life. Copyright © 2015 Elsevier Inc. All rights reserved.

Thiocoraline alters neuroendocrine phenotype and activates the Notch pathway in MTC-TT cell line

PubMed Central

Tesfazghi, Sara; Eide, Jacob; Dammalapati, Ajitha; Korlesky, Colin; Wyche, Thomas P; Bugni, Tim S; Chen, Herbert; Jaskula-Sztul, Renata

2013-01-01

Medullary thyroid cancer (MTC) is an aggressive neuroendocrine tumor (NET). Previous research has shown that activation of Notch signaling has a tumor suppressor role in NETs. The potential therapeutic effect of thiocoraline on the activation of the Notch pathway in an MTC cell line (TT) was investigated. Thiocoraline was isolated from a marine bacterium Verrucosispora sp. MTT assay (3-[4, 5-dimethylthiazole-2-yl]-2, 5-diphenyltetrazolium bromide) was used to determine the IC50 value and to measure cell proliferation. Western blot revealed the expression of Notch isoforms, NET, and cell cycle markers. Cell cycle progression was validated by flow cytometry. The mRNA expression of Notch isoforms and downstream targets were measured using real-time PCR. The IC50 value for thiocoraline treatment in TT cells was determined to be 7.6 nmol/L. Thiocoraline treatment decreased cell proliferation in a dose- and time-dependent manner. The mechanism of growth inhibition was found to be cell cycle arrest in G1 phase. Thiocoraline activated the Notch pathway as demonstrated by the dose-dependent increase in mRNA and protein expression of Notch isoforms. Furthermore, treatment with thiocoraline resulted in changes in the expression of downstream targets of the Notch pathway (HES1, HES2, HES6, HEY1, and HEY2) and reduced expression of NET markers, CgA, and ASCL1. Thiocoraline is a potent Notch pathway activator and an inhibitor of MTC-TT cell proliferation at low nanomolar concentrations. These results provide exciting evidence for the use of thiocoraline as a potential treatment for intractable MTC. Thiocoraline is a potent Notch pathway activator and an inhibitor of medullary thyroid cancer cell line (MTC-TT) cell proliferation at low nanomolar concentrations. These results provide evidence for the use of thiocoraline as a potential treatment for intractable MTC. PMID:24403239

Cannabinoids Δ9-Tetrahydrocannabinol and Cannabidiol Differentially Inhibit the Lipopolysaccharide-activated NF-κB and Interferon-β/STAT Proinflammatory Pathways in BV-2 Microglial Cells*

PubMed Central

Kozela, Ewa; Pietr, Maciej; Juknat, Ana; Rimmerman, Neta; Levy, Rivka; Vogel, Zvi

2010-01-01

Cannabinoids have been shown to exert anti-inflammatory activities in various in vivo and in vitro experimental models as well as ameliorate various inflammatory degenerative diseases. However, the mechanisms of these effects are not completely understood. Using the BV-2 mouse microglial cell line and lipopolysaccharide (LPS) to induce an inflammatory response, we studied the signaling pathways engaged in the anti-inflammatory effects of cannabinoids as well as their influence on the expression of several genes known to be involved in inflammation. We found that the two major cannabinoids present in marijuana, Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), decrease the production and release of proinflammatory cytokines, including interleukin-1β, interleukin-6, and interferon (IFN)β, from LPS-activated microglial cells. The cannabinoid anti-inflammatory action does not seem to involve the CB1 and CB2 cannabinoid receptors or the abn-CBD-sensitive receptors. In addition, we found that THC and CBD act through different, although partially overlapping, mechanisms. CBD, but not THC, reduces the activity of the NF-κB pathway, a primary pathway regulating the expression of proinflammatory genes. Moreover, CBD, but not THC, up-regulates the activation of the STAT3 transcription factor, an element of homeostatic mechanism(s) inducing anti-inflammatory events. Following CBD treatment, but less so with THC, we observed a decreased level of mRNA for the Socs3 gene, a main negative regulator of STATs and particularly of STAT3. However, both CBD and THC decreased the activation of the LPS-induced STAT1 transcription factor, a key player in IFNβ-dependent proinflammatory processes. In summary, our observations show that CBD and THC vary in their effects on the anti-inflammatory pathways, including the NF-κB and IFNβ-dependent pathways. PMID:19910459

Coordinate regulation of the Suf and Isc Fe-S cluster biogenesis pathways by IscR is essential for viability of Escherichia coli.

PubMed

Mettert, Erin L; Kiley, Patricia J

2014-12-01

Fe-S cluster biogenesis is essential for the viability of most organisms. In Escherichia coli, this process requires either the housekeeping Isc or the stress-induced Suf pathway. The global regulator IscR coordinates cluster synthesis by repressing transcription of the isc operon by [2Fe-2S]-IscR and activating expression of the suf operon. We show that either [2Fe-2S]-IscR or apo-IscR can activate suf, making expression sensitive to mainly IscR levels and not the cluster state, unlike isc expression. We also demonstrate that in the absence of isc, IscR-dependent suf activation is essential since strains lacking both the Isc pathway and IscR were not viable unless Suf was expressed ectopically. Similarly, removal of the IscR binding site in the sufA promoter also led to a requirement for isc. Furthermore, suf expression was increased in a Δisc mutant, presumably due to increased IscR levels in this mutant. This was surprising because the iron-dependent repressor Fur, whose higher-affinity binding at the sufA promoter should occlude IscR binding, showed only partial repression. In addition, Fur derepression was not sufficient for viability in the absence of IscR and the Isc pathway, highlighting the importance of direct IscR activation. Finally, a mutant lacking Fur and the Isc pathway increased suf expression to the highest observed levels and nearly restored [2Fe-2S]-IscR activity, providing a mechanism for regulating IscR activity under stress conditions. Together, these findings have enhanced our understanding of the homeostatic mechanism by which cells use one regulator, IscR, to differentially control Fe-S cluster biogenesis pathways to ensure viability. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Transcriptional Pathways Altered in Response to Vibration in a Model of Hand-Arm Vibration Syndrome

PubMed Central

Waugh, Stacey; Kashon, Michael L.; Li, Shengqiao; Miller, Gerome R.; Johnson, Claud; Krajnak, Kristine

2016-01-01

Objective The aim of this study was to use an established model of vibration-induced injury to assess frequency-dependent changes in transcript expression in skin, artery, and nerve tissues. Methods Transcript expression in tissues from control and vibration-exposed rats (4 h/day for 10 days at 62.5, 125, or 250 Hz; 49 m/s2, rms) was measured. Transcripts affected by vibration were used in bioinformatics analyses to identify molecular- and disease-related pathways associated with exposure to vibration. Results Analyses revealed that cancer-related pathways showed frequency-dependent changes in activation or inhibition. Most notably, the breast-related cancer-1 pathway was affected. Other pathways associated with breast cancer type 1 susceptibility protein related signaling, or associated with cancer and cell cycle/cell survivability were also affected. Conclusion Occupational exposure to vibration may result in DNA damage and alterations in cell signaling pathways that have significant effects on cellular division. PMID:27058473

β-Catenin-Dependent Wnt Signaling in C. elegans: Teaching an Old Dog a New Trick

PubMed Central

Jackson, Belinda M.; Eisenmann, David M.

2012-01-01

Wnt signaling is an evolutionarily ancient pathway used to regulate many events during metazoan development. Genetic results from Caenorhabditis elegans more than a dozen years ago suggested that Wnt signaling in this nematode worm might be different than in vertebrates and Drosophila: the worm had a small number of Wnts, too many β-catenins, and some Wnt pathway components functioned in an opposite manner than in other species. Work over the ensuing years has clarified that C. elegans does possess a canonical Wnt/β-catenin signaling pathway similar to that in other metazoans, but that the majority of Wnt signaling in this species may proceed via a variant Wnt/β-catenin signaling pathway that uses some new components (mitogen-activated protein kinase signaling enzymes), and in which some conserved pathway components (β-catenin, T-cell factor [TCF]) are used in new and interesting ways. This review summarizes our current understanding of the canonical and novel TCF/β-catenin-dependent signaling pathways in C. elegans. PMID:22745286

PARP1-mediated necrosis is dependent on parallel JNK and Ca2+/calpain pathways

PubMed Central

Douglas, Diana L.; Baines, Christopher P.

2014-01-01

ABSTRACT Poly(ADP-ribose) polymerase-1 (PARP1) is a nuclear enzyme that can trigger caspase-independent necrosis. Two main mechanisms for this have been proposed: one involving RIP1 and JNK kinases and mitochondrial permeability transition (MPT), the other involving calpain-mediated activation of Bax and mitochondrial release of apoptosis-inducing factor (AIF). However, whether these two mechanisms represent distinct pathways for PARP1-induced necrosis, or whether they are simply different components of the same pathway has yet to be tested. Mouse embryonic fibroblasts (MEFs) were treated with either N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) or β-Lapachone, resulting in PARP1-dependent necrosis. This was associated with increases in calpain activity, JNK activation and AIF translocation. JNK inhibition significantly reduced MNNG- and β-Lapachone-induced JNK activation, AIF translocation, and necrosis, but not calpain activation. In contrast, inhibition of calpain either by Ca2+ chelation or knockdown attenuated necrosis, but did not affect JNK activation or AIF translocation. To our surprise, genetic and/or pharmacological inhibition of RIP1, AIF, Bax and the MPT pore failed to abrogate MNNG- and β-Lapachone-induced necrosis. In conclusion, although JNK and calpain both contribute to PARP1-induced necrosis, they do so via parallel mechanisms. PMID:25052090

Novel mechanism of JNK pathway activation by adenoviral E1A

PubMed Central

Morrison, Helen; Pospelova, Tatiana V.; Pospelov, Valery A.; Herrlich, Peter

2014-01-01

The adenoviral oncoprotein E1A influences cellular regulation by interacting with a number of cellular proteins. In collaboration with complementary oncogenes, E1A fully transforms primary cells. As part of this action, E1A inhibits transcription of c-Jun:Fos target genes while promoting that of c-Jun:ATF2-dependent genes including jun. Both c-Jun and ATF2 are hyperphosphorylated in response to E1A. In the current study, E1A was fused with the ligand binding domain of the estrogen receptor (E1A-ER) to monitor the immediate effect of E1A activation. With this approach we now show that E1A activates c-Jun N-terminal kinase (JNK), the upstream kinases MKK4 and MKK7, as well as the small GTPase Rac1. Activation of the JNK pathway requires the N-terminal domain of E1A, and, importantly, is independent of transcription. In addition, it requires the presence of ERM proteins. Downregulation of signaling components upstream of JNK inhibits E1A-dependent JNK/c-Jun activation. Taking these findings together, we show that E1A activates the JNK/c-Jun signaling pathway upstream of Rac1 in a transcription-independent manner, demonstrating a novel mechanism of E1A action. PMID:24742962

Rhodococcus erythropolis DCL14 Contains a Novel Degradation Pathway for Limonene

PubMed Central

van der Werf, Mariët J.; Swarts, Henk J.; de Bont, Jan A. M.

1999-01-01

Strain DCL14, which is able to grow on limonene as a sole source of carbon and energy, was isolated from a freshwater sediment sample. This organism was identified as a strain of Rhodococcus erythropolis by chemotaxonomic and genetic studies. R. erythropolis DCL14 also assimilated the terpenes limonene-1,2-epoxide, limonene-1,2-diol, carveol, carvone, and (−)-menthol, while perillyl alcohol was not utilized as a carbon and energy source. Induction tests with cells grown on limonene revealed that the oxygen consumption rates with limonene-1,2-epoxide, limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and carveol were high. Limonene-induced cells of R. erythropolis DCL14 contained the following four novel enzymatic activities involved in the limonene degradation pathway of this microorganism: a flavin adenine dinucleotide- and NADH-dependent limonene 1,2-monooxygenase activity, a cofactor-independent limonene-1,2-epoxide hydrolase activity, a dichlorophenolindophenol-dependent limonene-1,2-diol dehydrogenase activity, and an NADPH-dependent 1-hydroxy-2-oxolimonene 1,2-monooxygenase activity. Product accumulation studies showed that (1S,2S,4R)-limonene-1,2-diol, (1S,4R)-1-hydroxy-2-oxolimonene, and (3R)-3-isopropenyl-6-oxoheptanoate were intermediates in the (4R)-limonene degradation pathway. The opposite enantiomers [(1R,2R,4S)-limonene-1,2-diol, (1R,4S)-1-hydroxy-2-oxolimonene, and (3S)-3-isopropenyl-6-oxoheptanoate] were found in the (4S)-limonene degradation pathway, while accumulation of (1R,2S,4S)-limonene-1,2-diol from (4S)-limonene was also observed. These results show that R. erythropolis DCL14 metabolizes both enantiomers of limonene via a novel degradation pathway that starts with epoxidation at the 1,2 double bond forming limonene-1,2-epoxide. This epoxide is subsequently converted to limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and 7-hydroxy-4-isopropenyl-7-methyl-2-oxo-oxepanone. This lactone spontaneously rearranges to form 3-isopropenyl-6-oxoheptanoate. In the presence of coenzyme A and ATP this acid is converted further, and this finding, together with the high levels of isocitrate lyase activity in extracts of limonene-grown cells, suggests that further degradation takes place via the β-oxidation pathway. PMID:10224006

Regulatory pathways in the European Union.

PubMed

Kohler, Manuela

2011-01-01

In principle, there are three defined procedures to obtain approval for a medicinal product in the European Union. As discussed in this overview of the procedures, the decision on which regulatory pathway to use will depend on the nature of the active substance, the target indication(s), the history of product and/or the marketing strategy.

GPCR-mediated PLCβγ/PKCβ/PKD signaling pathway regulates the cofilin phosphatase slingshot 2 in neutrophil chemotaxis

PubMed Central

Xu, Xuehua; Gera, Nidhi; Li, Hongyan; Yun, Michelle; Zhang, Liyong; Wang, Youhong; Wang, Q. Jane; Jin, Tian

2015-01-01

Chemotaxis requires precisely coordinated polymerization and depolymerization of the actin cytoskeleton at leading fronts of migrating cells. However, GPCR activation-controlled F-actin depolymerization remains largely elusive. Here, we reveal a novel signaling pathway, including Gαi, PLC, PKCβ, protein kinase D (PKD), and SSH2, in control of cofilin phosphorylation and actin cytoskeletal reorganization, which is essential for neutrophil chemotaxis. We show that PKD is essential for neutrophil chemotaxis and that GPCR-mediated PKD activation depends on PLC/PKC signaling. More importantly, we discover that GPCR activation recruits/activates PLCγ2 in a PI3K-dependent manner. We further verify that PKCβ specifically interacts with PKD1 and is required for chemotaxis. Finally, we identify slingshot 2 (SSH2), a phosphatase of cofilin (actin depolymerization factor), as a target of PKD1 that regulates cofilin phosphorylation and remodeling of the actin cytoskeleton during neutrophil chemotaxis. PMID:25568344

Reciprocal feedback regulation of PI3K and androgen receptor signaling in PTEN-deficient prostate cancer

PubMed Central

Carver, Brett S; Chapinski, Caren; Wongvipat, John; Hieronymus, Haley; Chen, Yu; Chandarlapaty, Sarat; Arora, Vivek K; Le, Carl; Koutcher, Jason; Scher, Howard; Scardino, Peter T; Rosen, Neal; Sawyers, Charles L

2011-01-01

Summary Prostate cancer is characterized by its dependence on androgen receptor and frequent activation of PI3K signaling. We find that AR transcriptional output is decreased in human and murine tumors with PTEN deletion and that PI3K pathway inhibition activates AR signaling by relieving feedback inhibition of HER kinases. Similarly, AR inhibition activates AKT signaling by reducing levels of the AKT phosphatase PHLPP. Thus, these two oncogenic pathways cross-regulate each other by reciprocal feedback. Inhibition of one activates the other, thereby maintaining tumor cell survival. However, combined pharmacologic inhibition of PI3K and AR signaling caused near complete prostate cancer regressions in a Pten-deficient murine prostate cancer model and in human prostate cancer xenografts, indicating that both pathways coordinately support survival. Significance The two most frequently activated signaling pathways in prostate cancer are driven by AR and PI3K. Inhibitors of the PI3K pathway are in early clinical trials and AR inhibitors confer clinical responses in most patients. However, these inhibitors rarely induce tumor regression in preclinical models. Here we show that these pathways regulate each other by reciprocal negative feedback, such that inhibition of one activates the other. Therefore, tumor cells can adapt and survive when either single pathway is inhibited pharmacologically. Our demonstration of profound tumor regressions with combined pathway inhibition in preclinical prostate tumor models provides rationale for combination therapy in patients. PMID:21575859

Carvedilol, a third-generation β-blocker prevents oxidative stress-induced neuronal death and activates Nrf2/ARE pathway in HT22 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ouyang, Ying; Chen, Ziwei; Tan, Min

Highlights: •Carvedilol significantly prevented oxidative stress-induced cell death. •Carvedilol significantly decreased the production of ROS. •Carvedilol activated Nrf2/ARE pathway. •Carvedilol increased the protein levels of HO-1 and NQO-1. -- Abstract: Carvedilol, a nonselective β-adrenoreceptor blocker with pleiotropic activities has been shown to exert neuroprotective effect due to its antioxidant property. However, the neuroprotective mechanism of carvedilol is still not fully uncovered. Nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway is an important cellular stress response pathway involved in neuroprotection. Here we investigated the effect of carvedilol on oxidative stress-induced cell death (glutamate 2 mM and H{sub 2}O{sub 2}more » 600 μM) and the activity of Nrf2/ARE pathway in HT22 hippocampal cells. Carvedilol significantly increased cell viability and decreased ROS in HT22 cells exposed to glutamate or H{sub 2}O{sub 2}. Furthermore, carvedilol activated the Nrf2/ARE pathway in a concentration-dependent manner, and increased the protein levels of heme oxygenase-1(HO-1) and NAD(P)H quinone oxidoreductase-1(NQO-1), two downstream factors of the Nrf2/ARE pathway. Collectively, our results indicate that carvedilol protects neuronal cell against glutamate- and H{sub 2}O{sub 2}-induced neurotoxicity possibly through activating the Nrf2/ARE signaling pathway.« less

Noncanonical transforming growth factor β signaling in scleroderma fibrosis

PubMed Central

Trojanowska, Maria

2014-01-01

Purpose of review Persistent transforming growth factor β (TGF-β) signaling is the major factor contributing to scleroderma (SSc) fibrosis. This review will summarize recent progress on the noncanonical TGF-β signaling pathways and their role in SSc fibrosis. Recent findings Canonical TGF-β signaling involves activation of the TGF-β receptors and downstream signal transducers Smad2/3. The term noncanonical TGF-β signaling includes a variety of intracellular signaling pathways activated by TGF-β independently of Smad2/3 activation. There is evidence that these pathways play important role in SSc fibrosis. In a subset of SSc fibroblasts, a multiligand receptor complex consisting of TGF-β and CCN2 receptors drives constitutive activation of the Smad1 pathway. CCN2 is also a primary effector of this pathway, thus establishing an autocrine loop that amplifies TGF-β signaling. SSc fibroblasts also demonstrate reduced expression of endogenous antagonists of TGF-β signaling including transcriptional repressors, Friend leukemia integration-1 and perixosome proliferator-activated receptor-γ, as well as inhibitor of Smad3 phosphorylation, PTEN. PTEN is a key mediator of the cross-talk between the sphingosine kinase and the TGF-β pathways. Summary Discovery of the role of noncanonical TGF-β signaling in fibrosis offers new molecular targets for the antifibrotic therapies. Due to the heterogeneous nature of SSc, knowledge of these pathways could help to tailor the therapy to the individual patient depending on the activation status of a specific profibrotic pathway. PMID:19713852

Sonic Hedgehog modulates EGFR dependent proliferation of neural stem cells during late mouse embryogenesis through EGFR transactivation

PubMed Central

Reinchisi, Gisela; Parada, Margarita; Lois, Pablo; Oyanadel, Claudia; Shaughnessy, Ronan; Gonzalez, Alfonso; Palma, Verónica

2013-01-01

Sonic Hedgehog (Shh/GLI) and EGFR signaling pathways modulate Neural Stem Cell (NSC) proliferation. How these signals cooperate is therefore critical for understanding normal brain development and function. Here we report a novel acute effect of Shh signaling on EGFR function. We show that during late neocortex development, Shh mediates the activation of the ERK1/2 signaling pathway in Radial Glial cells (RGC) through EGFR transactivation. This process is dependent on metalloprotease activity and accounts for almost 50% of the EGFR-dependent mitogenic response of late NSCs. Furthermore, in HeLa cancer cells, a well-known model for studying the EGFR receptor function, Shh also induces cell proliferation involving EGFR activation, as reflected by EGFR internalization and ERK1/2 phosphorylation. These findings may have important implications for understanding the mechanisms that regulate NSC proliferation during neurogenesis and may lead to novel approaches to the treatment of tumors. PMID:24133411

Activation of PTHrP-cAMP-CREB1 signaling following p53 loss is essential for osteosarcoma initiation and maintenance.

PubMed

Walia, Mannu K; Ho, Patricia Mw; Taylor, Scott; Ng, Alvin Jm; Gupte, Ankita; Chalk, Alistair M; Zannettino, Andrew Cw; Martin, T John; Walkley, Carl R

2016-04-12

Mutations in the P53 pathway are a hallmark of human cancer. The identification of pathways upon which p53-deficient cells depend could reveal therapeutic targets that may spare normal cells with intact p53. In contrast to P53 point mutations in other cancer, complete loss of P53 is a frequent event in osteosarcoma (OS), the most common cancer of bone. The consequences of p53 loss for osteoblastic cells and OS development are poorly understood. Here we use murine OS models to demonstrate that elevated Pthlh (Pthrp), cAMP levels and signalling via CREB1 are characteristic of both p53-deficient osteoblasts and OS. Normal osteoblasts survive depletion of both PTHrP and CREB1. In contrast, p53-deficient osteoblasts and OS depend upon continuous activation of this pathway and undergo proliferation arrest and apoptosis in the absence of PTHrP or CREB1. Our results identify the PTHrP-cAMP-CREB1 axis as an attractive pathway for therapeutic inhibition in OS.

Hippo, TGF-β, and Src-MAPK pathways regulate transcription of the upd3 cytokine in Drosophila enterocytes upon bacterial infection.

PubMed

Houtz, Philip; Bonfini, Alessandro; Liu, Xi; Revah, Jonathan; Guillou, Aurélien; Poidevin, Mickael; Hens, Korneel; Huang, Hsin-Yi; Deplancke, Bart; Tsai, Yu-Chen; Buchon, Nicolas

2017-11-01

Cytokine signaling is responsible for coordinating conserved epithelial regeneration and immune responses in the digestive tract. In the Drosophila midgut, Upd3 is a major cytokine, which is induced in enterocytes (EC) and enteroblasts (EB) upon oral infection, and initiates intestinal stem cell (ISC) dependent tissue repair. To date, the genetic network directing upd3 transcription remains largely uncharacterized. Here, we have identified the key infection-responsive enhancers of the upd3 gene and show that distinct enhancers respond to various stresses. Furthermore, through functional genetic screening, bioinformatic analyses and yeast one-hybrid screening, we determined that the transcription factors Scalloped (Sd), Mothers against dpp (Mad), and D-Fos are principal regulators of upd3 expression. Our study demonstrates that upd3 transcription in the gut is regulated by the activation of multiple pathways, including the Hippo, TGF-β/Dpp, and Src, as well as p38-dependent MAPK pathways. Thus, these essential pathways, which are known to control ISC proliferation cell-autonomously, are also activated in ECs to promote tissue turnover the regulation of upd3 transcription.

Dual pathways to prospective remembering

PubMed Central

McDaniel, Mark A.; Umanath, Sharda; Einstein, Gilles O.; Waldum, Emily R.

2015-01-01

According to the multiprocess framework (McDaniel and Einstein, 2000), the cognitive system can support prospective memory (PM) retrieval through two general pathways. One pathway depends on top–down attentional control processes that maintain activation of the intention and/or monitor the environment for the triggering or target cues that indicate that the intention should be executed. A second pathway depends on (bottom–up) spontaneous retrieval processes, processes that are often triggered by a PM target cue; critically, spontaneous retrieval is assumed not to require monitoring or active maintenance of the intention. Given demand characteristics associated with experimental settings, however, participants are often inclined to monitor, thereby potentially masking discovery of bottom–up spontaneous retrieval processes. In this article, we discuss parameters of laboratory PM paradigms to discourage monitoring and review recent behavioral evidence from such paradigms that implicate spontaneous retrieval in PM. We then re-examine the neuro-imaging evidence from the lens of the multiprocess framework and suggest some critical modifications to existing neuro-cognitive interpretations of the neuro-imaging results. These modifications illuminate possible directions and refinements for further neuro-imaging investigations of PM. PMID:26236213

Wnt signaling during tooth replacement in zebrafish (Danio rerio): pitfalls and perspectives

PubMed Central

Huysseune, Ann; Soenens, Mieke; Elderweirdt, Fien

2014-01-01

The canonical (β-catenin dependent) Wnt signaling pathway has emerged as a likely candidate for regulating tooth replacement in continuously renewing dentitions. So far, the involvement of canonical Wnt signaling has been experimentally demonstrated predominantly in amniotes. These studies tend to show stimulation of tooth formation by activation of the Wnt pathway, and inhibition of tooth formation when blocking the pathway. Here, we report a strong and dynamic expression of the soluble Wnt inhibitor dickkopf1 (dkk1) in developing zebrafish (Danio rerio) tooth germs, suggesting an active repression of Wnt signaling during morphogenesis and cytodifferentiation of a tooth, and derepression of Wnt signaling during start of replacement tooth formation. To further analyse the role of Wnt signaling, we used different gain-of-function approaches. These yielded disjunct results, yet none of them indicating enhanced tooth replacement. Thus, masterblind (mbl) mutants, defective in axin1, mimic overexpression of Wnt, but display a normally patterned dentition in which teeth are replaced at the appropriate times and positions. Activating the pathway with LiCl had variable outcomes, either resulting in the absence, or the delayed formation, of first-generation teeth, or yielding a regular dentition with normal replacement, but no supernumerary teeth or accelerated tooth replacement. The failure so far to influence tooth replacement in the zebrafish by perturbing Wnt signaling is discussed in the light of (i) potential technical pitfalls related to dose- or time-dependency, (ii) the complexity of the canonical Wnt pathway, and (iii) species-specific differences in the nature and activity of pathway components. Finally, we emphasize the importance of in-depth knowledge of the wild-type pattern for reliable interpretations. It is hoped that our analysis can be inspiring to critically assess and elucidate the role of Wnt signaling in tooth development in polyphyodonts. PMID:25339911

A Novel Role for the BMP Antagonist Noggin in Sensitizing Cells to Non-canonical Wnt-5a/Ror2/Disheveled Pathway Activation

PubMed Central

Bernatik, Ondrej; Radaszkiewicz, Tomasz; Behal, Martin; Dave, Zankruti; Witte, Florian; Mahl, Annika; Cernohorsky, Nicole H.; Krejci, Pavel; Stricker, Sigmar; Bryja, Vitezslav

2017-01-01

Mammalian limb development is driven by the integrative input from several signaling pathways; a failure to receive or a misinterpretation of these signals results in skeletal defects. The brachydactylies, a group of overlapping inherited human hand malformation syndromes, are mainly caused by mutations in BMP signaling pathway components. Two closely related forms, Brachydactyly type B2 (BDB2) and BDB1 are caused by mutations in the BMP antagonist Noggin (NOG) and the atypical receptor tyrosine kinase ROR2 that acts as a receptor in the non-canonical Wnt pathway. Genetic analysis of Nog and Ror2 functional interaction via crossing Noggin and Ror2 mutant mice revealed a widening of skeletal elements in compound but not in any of the single mutants, thus indicating genetic interaction. Since ROR2 is a non-canonical Wnt co-receptor specific for Wnt-5a we speculated that this phenotype might be a result of deregulated Wnt-5a signaling activation, which is known to be essential for limb skeletal elements growth and patterning. We show that Noggin potentiates activation of the Wnt-5a-Ror2-Disheveled (Dvl) pathway in mouse embryonic fibroblast (MEF) cells in a Ror2-dependent fashion. Rat chondrosarcoma chondrocytes (RCS), however, are not able to respond to Noggin in this fashion unless growth arrest is induced by FGF2. In summary, our data demonstrate genetic interaction between Noggin and Ror2 and show that Noggin can sensitize cells to Wnt-5a/Ror2-mediated non-canonical Wnt signaling, a feature that in cartilage may depend on the presence of active FGF signaling. These findings indicate an unappreciated function of Noggin that will help to understand BMP and Wnt/PCP signaling pathway interactions. PMID:28523267

Regulation of mGlu4 metabotropic glutamate receptor signaling by type-2 G-protein coupled receptor kinase (GRK2).

PubMed

Iacovelli, L; Capobianco, L; Iula, M; Di Giorgi Gerevini, V; Picascia, A; Blahos, J; Melchiorri, D; Nicoletti, F; De Blasi, A

2004-05-01

We examined the role of G-protein coupled receptor kinase-2 (GRK2) in the homologous desensitization of mGlu4 metabotropic glutamate receptors transiently expressed in human embryonic kidney (HEK) 293 cells. Receptor activation with the agonist l-2-amino-4-phosphonobutanoate (l-AP4) stimulated at least two distinct signaling pathways: inhibition of cAMP formation and activation of the mitogen-activated protein kinase (MAPK) pathway [assessed by Western blot analysis of phosphorylated extracellular signal-regulated kinase (ERK) 1 and 2]. Activation of both pathways was attenuated by pertussis toxin. Overexpression of GRK2 (but not GRK4) largely attenuated the stimulation of the MAPK pathway by l-AP4, whereas it slightly potentiated the inhibition of FSK-stimulated cAMP formation. Transfection with a kinase-dead mutant of GRK2 (GRK2-K220R) or with the C-terminal fragment of GRK2 also reduced the mGlu4-mediated stimulation of MAPK, suggesting that GRK2 binds to the Gbetagamma subunits to inhibit signal propagation toward the MAPK pathway. This was confirmed by the evidence that GRK2 coimmunoprecipitated with Gbetagamma subunits in an agonist-dependent manner. Finally, neither GRK2 nor its kinase-dead mutant had any effect on agonist-induced mGlu4 receptor internalization in HEK293 cells transiently transfected with GFP-tagged receptors. Agonist-dependent internalization was instead abolished by a negative-dominant mutant of dynamin, which also reduced the stimulation of MAPK pathway by l-AP4. We speculate that GRK2 acts as a "switch molecule" by inhibiting the mGlu4 receptor-mediated stimulation of MAPK and therefore directing the signal propagation toward the inhibition of adenylyl cyclase.

Human osteocalcin and bone sialoprotein mediating osteomimicry of prostate cancer cells: role of cAMP-dependent protein kinase A signaling pathway.

PubMed

Huang, Wen-Chin; Xie, Zhihui; Konaka, Hiroyuki; Sodek, Jaro; Zhau, Haiyen E; Chung, Leland W K

2005-03-15

Osteocalcin and bone sialoprotein are the most abundant noncollagenous bone matrix proteins expressed by osteoblasts. Surprisingly, osteocalcin and bone sialoprotein are also expressed by malignant but not normal prostate epithelial cells. The purpose of this study is to investigate how osteocalcin and bone sialoprotein expression is regulated in prostate cancer cells. Our investigation revealed that (a) human osteocalcin and bone sialoprotein promoter activities in an androgen-independent prostate cancer cell line of LNCaP lineage, C4-2B, were markedly enhanced 7- to 12-fold in a concentration-dependent manner by conditioned medium collected from prostate cancer and bone stromal cells. (b) Deletion analysis of human osteocalcin and bone sialoprotein promoter regions identified cyclic AMP (cAMP)-responsive elements (CRE) as the critical determinants for conditioned medium-mediated osteocalcin and bone sialoprotein gene expression in prostate cancer cells. Consistent with these results, the protein kinase A (PKA) pathway activators forskolin and dibutyryl cAMP and the PKA pathway inhibitor H-89, respectively, increased or repressed human osteocalcin and bone sialoprotein promoter activities. (c) Electrophoretic mobility shift assay showed that conditioned medium-mediated stimulation of human osteocalcin and bone sialoprotein promoter activities occurs through increased interaction between CRE and CRE-binding protein. (d) Conditioned medium was found to induce human osteocalcin and bone sialoprotein promoter activities via increased CRE/CRE-binding protein interaction in a cell background-dependent manner, with marked stimulation in selected prostate cancer but not bone stromal cells. Collectively, these results suggest that osteocalcin and bone sialoprotein expression is coordinated and regulated through cAMP-dependent PKA signaling, which may define the molecular basis of the osteomimicry exhibited by prostate cancer cells.

Oxaloacetate supplementation increases lifespan in Caenorhabditis elegans through an AMPK/FOXO-dependent pathway

PubMed Central

Williams, David S.; Cash, Alan; Hamadani, Lara; Diemer, Tanja

2010-01-01

Summary Reduced dietary intake increases lifespan in a wide variety of organisms. It also retards disease progression. We tested whether dietary supplementation of citric acid cycle metabolites could mimic this lifespan effect. We report that oxaloacetate supplementation increased lifespan in Caenorhabditis elegans. The increase was dependent on the transcription factor, FOXO/DAF-16, and the energy sensor, AMP-activated protein kinase, indicating involvement of a pathway that is also required for lifespan extension through dietary restriction. These results demonstrate that supplementation of the citric acid cycle metabolite, oxaloacetate, influences a longevity pathway, and suggest a tractable means of introducing the health-related benefits of dietary restriction. PMID:19793063

Cross Talk between Nucleotide Synthesis Pathways with Cellular Immunity in Constraining Hepatitis E Virus Replication

PubMed Central

Wang, Yijin; Wang, Wenshi; Xu, Lei; Zhou, Xinying; Shokrollahi, Ehsan; Felczak, Krzysztof; van der Laan, Luc J. W.; Pankiewicz, Krzysztof W.; Sprengers, Dave; Raat, Nicolaas J. H.; Metselaar, Herold J.; Peppelenbosch, Maikel P.

2016-01-01

Viruses are solely dependent on host cells to propagate; therefore, understanding virus-host interaction is important for antiviral drug development. Since de novo nucleotide biosynthesis is essentially required for both host cell metabolism and viral replication, specific catalytic enzymes of these pathways have been explored as potential antiviral targets. In this study, we investigated the role of different enzymatic cascades of nucleotide biosynthesis in hepatitis E virus (HEV) replication. By profiling various pharmacological inhibitors of nucleotide biosynthesis, we found that targeting the early steps of the purine biosynthesis pathway led to the enhancement of HEV replication, whereas targeting the later step resulted in potent antiviral activity via the depletion of purine nucleotide. Furthermore, the inhibition of the pyrimidine pathway resulted in potent anti-HEV activity. Interestingly, all of these inhibitors with anti-HEV activity concurrently triggered the induction of antiviral interferon-stimulated genes (ISGs). Although ISGs are commonly induced by interferons via the JAK-STAT pathway, their induction by nucleotide synthesis inhibitors is completely independent of this classical mechanism. In conclusion, this study revealed an unconventional novel mechanism of cross talk between nucleotide biosynthesis pathways and cellular antiviral immunity in constraining HEV infection. Targeting particular enzymes in nucleotide biosynthesis represents a viable option for antiviral drug development against HEV. HEV is the most common cause of acute viral hepatitis worldwide and is also associated with chronic hepatitis, especially in immunocompromised patients. Although often an acute and self-limiting infection in the general population, HEV can cause severe morbidity and mortality in certain patients, a problem compounded by the lack of FDA-approved anti-HEV medication available. In this study, we have investigated the role of the nucleotide synthesis pathway in HEV infection and its potential for antiviral drug development. We show that targeting the later but not the early steps of the purine synthesis pathway exerts strong anti-HEV activity. In particular, IMP dehydrogenase (IMPDH) is the most important anti-HEV target of this cascade. Importantly, the clinically used IMPDH inhibitors, including mycophenolic acid and ribavirin, have potent anti-HEV activity. Furthermore, targeting the pyrimidine synthesis pathway also exerts potent antiviral activity against HEV. Interestingly, antiviral effects of nucleotide synthesis pathway inhibitors appear to depend on the medication-induced transcription of antiviral interferon-stimulated genes. Thus, this study reveals an unconventional novel mechanism as to how nucleotide synthesis pathway inhibitors can counteract HEV replication. PMID:26926637

A rapid and transient ROS generation by cadmium triggers apoptosis via caspase-dependent pathway in HepG2 cells and this is inhibited through N-acetylcysteine-mediated catalase upregulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oh, Seon-Hee; Lim, Sung-Chul

2006-05-01

Although reactive oxygen species (ROS) have been implicated in cadmium (Cd)-induced hepatotoxicity, the role of ROS in this pathway remains unclear. Therefore, we attempted to determine the molecular mechanisms relevant to Cd-induced cell death in HepG2 cells. Cd was found to induce apoptosis in the HepG2 cells in a time- and dose-dependent fashion, as confirmed by DNA fragmentation analysis and TUNEL staining. In the early stages, both rapid and transient ROS generation triggered apoptosis via Fas activation and subsequent caspase-8-dependent Bid cleavage, as well as by calpain-mediated mitochondrial Bax cleavage. The timing of Bid activation was coincided with the timingmore » at which the mitochondrial transmembrane potential (MMP) collapsed as well as the cytochrome c (Cyt c) released into the cytosol. Furthermore, mitochondrial permeability transition (MPT) pore inhibitors, such as cyclosporin A (CsA) and bongkrekic acid (BA), did not block Cd-induced ROS generation, MMP collapse and Cyt c release. N-acetylcysteine (NAC) pretreatment resulted in the complete inhibition of the Cd-induced apoptosis via catalase upregulation and subsequent Fas downregulation. NAC treatment also completely blocked the Cd-induced intracellular ROS generation, MMP collapse and Cyt c release, indicating that Cd-induced mitochondrial dysfunction may be regulated indirectly by ROS-mediated signaling pathway. Taken together, a rapid and transient ROS generation by Cd triggers apoptosis via caspase-dependent pathway and subsequent mitochondrial pathway. NAC inhibits Cd-induced apoptosis through the blocking of ROS generation as well as the catalase upregulation.« less

Probiotic-fermented purple sweet potato yogurt activates compensatory IGF‑IR/PI3K/Akt survival pathways and attenuates cardiac apoptosis in the hearts of spontaneously hypertensive rats.

PubMed

Lin, Pei-Pei; Hsieh, You-Miin; Kuo, Wei-Wen; Lin, Yueh-Min; Yeh, Yu-Lan; Lin, Chien-Chung; Tsai, Fuu-Jen; Tsai, Chang-Hai; Huang, Chih-Yang; Tsai, Cheng-Chih

2013-12-01

Apoptosis is recognized as a predictor of adverse outcomes in subjects with cardiac diseases. The aim of this study was to explore the effects of probiotic-fermented purple sweet potato yogurt (PSPY) with high γ-aminobutyric acid (GABA) content on cardiac apoptosis in spontaneously hypertensive rat (SHR) hearts. The rats were orally adminsitered with 2 different concentrations of PSPY (10 and 100%) or captopril, 15.6 mg/kg, body weight (BW)/day. The control group was administered distilled water. DAPI and TUNEL staining were used to detect the numbers of apoptotic cells. A decrease in the number of TUNEL-positive cardiac myocytes was observed in the SHR-PSPY (10 and 100%) groups. In addition, the levels of key components of the Fas receptor- and mitochondrial-dependent apoptotic pathways were determined by western blot analysis. The results revealed that the levels of the key components of the Fas receptor- and mitochondrial-dependent apoptotic pathway were significantly decreased in the SHR-captopril, and 10 and 100% PSPY groups. Additionally, the levels of phosphorylated insulin-like growth factor‑I receptor (p-IGF‑IR) were increased in SHR hearts from the SHR-control group; however, no recovery in the levels of downstream signaling components was observed. In addition, the levels of components of the compensatory IGF-IR-dependent survival pathway (p-PI3K and p-Akt) were all highly enhanced in the left ventricles in the hearts form the SHR-10 and 100% PSPY groups. Therefore, the oral administration of PSPY may attenuate cardiomyocyte apoptosis in SHR hearts by activating IGF‑IR-dependent survival signaling pathways.

Both live and dead Enterococci activate Caenorhabditis elegans host defense via immune and stress pathways.

PubMed

Yuen, Grace J; Ausubel, Frederick M

2018-12-31

The innate immune response of the nematode Caenorhabditis elegans has been extensively studied and a variety of Toll-independent immune response pathways have been identified. Surprisingly little, however, is known about how pathogens activate the C. elegans immune response. Enterococcus faecalis and Enterococcus faecium are closely related enterococcal species that exhibit significantly different levels of virulence in C. elegans infection models. Previous work has shown that activation of the C. elegans immune response by Pseudomonas aeruginosa involves P. aeruginosa-mediated host damage. Through ultrastructural imaging, we report that infection with either E. faecalis or E. faecium causes the worm intestine to become distended with proliferating bacteria in the absence of extensive morphological changes and apparent physical damage. Genetic analysis, whole-genome transcriptional profiling, and multiplexed gene expression analysis demonstrate that both enterococcal species, whether live or dead, induce a rapid and similar transcriptional defense response dependent upon previously described immune signaling pathways. The host response to E. faecium shows a stricter dependence upon stress response signaling pathways than the response to E. faecalis. Unexpectedly, we find that E. faecium is a C. elegans pathogen and that an active wild-type host defense response is required to keep an E. faecium infection at bay. These results provide new insights into the mechanisms underlying the C. elegans immune response to pathogen infection.

Focal adhesion kinase dependent activation of the PI3 kinase pathway by the functional soluble form of neurotensin receptor-3 in HT29 cells.

PubMed

Massa, Fabienne; Devader, Christelle; Béraud-Dufour, Sophie; Brau, Frédéric; Coppola, Thierry; Mazella, Jean

2013-05-01

The neurotensin (NT) receptor-3 (NTSR3), also called sortilin, is thought to display several functions including a role as a receptor or a co-receptor, in the sorting to plasma membrane and to lysosomes, and in the regulated secretion. The aim of this study was to investigate the function of the soluble form of NTSR3 (sNTSR3) released from several cell lines including colonic cancer cells. The human adenocarcinoma epithelial cell line HT29 has been used to monitor the release, the binding and internalization of sNTSR3 by radioreceptor assays and confocal microscopy. The modulation of the intracellular signaling pathways by the protein has been investigated by using Fura-2 fluorescence calcium imaging microscopy and Western blots analysis. We demonstrated that sNTSR3 specifically binds and internalizes into HT29 cells. This binding, independent from the transactivation of the epidermal growth factor receptor, leads to the increase of intracellular calcium concentration and to the activation of a FAK/Src-dependent activation of the PI3 kinase pathway. In conclusion, sNTSR3 released from the membrane bound NTSR3 is a functional protein able to activate intracellular pathways involved in cell survival but probably not in cell growth. Copyright © 2013 Elsevier Ltd. All rights reserved.

Survival of Human Multiple Myeloma Cells Is Dependent on MUC1 C-Terminal Transmembrane Subunit Oncoprotein Function

PubMed Central

Yin, Li; Ahmad, Rehan; Kosugi, Michio; Kufe, Turner; Vasir, Baldev; Avigan, David; Kharbanda, Surender

2010-01-01

The MUC1 C-terminal transmembrane subunit (MUC1-C) oncoprotein is a direct activator of the canonical nuclear factor-κB (NF-κB) RelA/p65 pathway and is aberrantly expressed in human multiple myeloma cells. However, it is not known whether multiple myeloma cells are sensitive to the disruption of MUC1-C function for survival. The present studies demonstrate that peptide inhibitors of MUC1-C oligomerization block growth of human multiple myeloma cells in vitro. Inhibition of MUC1-C function also blocked the interaction between MUC1-C and NF-κB p65 and activation of the NF-κB pathway. In addition, inhibition of MUC1-C in multiple myeloma cells was associated with activation of the intrinsic apoptotic pathway and induction of late apoptosis/necrosis. Primary multiple myeloma cells, but not normal B-cells, were also sensitive to MUC1-C inhibition. Significantly, treatment of established U266 multiple myeloma xenografts growing in nude mice with a lead candidate MUC1-C inhibitor resulted in complete tumor regression and lack of recurrence. These findings indicate that multiple myeloma cells are dependent on intact MUC1-C function for constitutive activation of the canonical NF-κB pathway and for their growth and survival. PMID:20444960

Both live and dead Enterococci activate Caenorhabditis elegans host defense via immune and stress pathways

PubMed Central

2018-01-01

ABSTRACT The innate immune response of the nematode Caenorhabditis elegans has been extensively studied and a variety of Toll-independent immune response pathways have been identified. Surprisingly little, however, is known about how pathogens activate the C. elegans immune response. Enterococcus faecalis and Enterococcus faecium are closely related enterococcal species that exhibit significantly different levels of virulence in C. elegans infection models. Previous work has shown that activation of the C. elegans immune response by Pseudomonas aeruginosa involves P. aeruginosa-mediated host damage. Through ultrastructural imaging, we report that infection with either E. faecalis or E. faecium causes the worm intestine to become distended with proliferating bacteria in the absence of extensive morphological changes and apparent physical damage. Genetic analysis, whole-genome transcriptional profiling, and multiplexed gene expression analysis demonstrate that both enterococcal species, whether live or dead, induce a rapid and similar transcriptional defense response dependent upon previously described immune signaling pathways. The host response to E. faecium shows a stricter dependence upon stress response signaling pathways than the response to E. faecalis. Unexpectedly, we find that E. faecium is a C. elegans pathogen and that an active wild-type host defense response is required to keep an E. faecium infection at bay. These results provide new insights into the mechanisms underlying the C. elegans immune response to pathogen infection. PMID:29436902

The signaling pathways by which the Fas/FasL system accelerates oocyte aging.

PubMed

Zhu, Jiang; Lin, Fei-Hu; Zhang, Jie; Lin, Juan; Li, Hong; Li, You-Wei; Tan, Xiu-Wen; Tan, Jing-He

2016-02-01

In spite of great efforts, the mechanisms for postovulatory oocyte aging are not fully understood. Although our previous work showed that the FasL/Fas signaling facilitated oocyte aging, the intra-oocyte signaling pathways are unknown. Furthermore, the mechanisms by which oxidative stress facilitates oocyte aging and the causal relationship between Ca2+ rises and caspase-3 activation and between the cell cycle and apoptosis during oocyte aging need detailed investigations. Our aim was to address these issues by studying the intra-oocyte signaling pathways for Fas/FasL to accelerate oocyte aging. The results indicated that sFasL released by cumulus cells activated Fas on the oocyte by increasing reactive oxygen species via activating NADPH oxidase. The activated Fas triggered Ca2+ release from the endoplasmic reticulum by activating phospholipase C-γ pathway and cytochrome c pathway. The cytoplasmic Ca2+ rises activated calcium/calmodulin-dependent protein kinase II (CaMKII) and caspase-3. While activated CaMKII increased oocyte susceptibility to activation by inactivating maturation-promoting factor (MPF) through cyclin B degradation, the activated caspase-3 facilitated further Ca2+releasing that activates more caspase-3 leading to oocyte fragmentation. Furthermore, caspase-3 activation and fragmentation were prevented in oocytes with a high MPF activity, suggesting that an oocyte must be in interphase to undergo apoptosis.

10′(Z),13′(E)-Heptadecadienylhydroquinone Inhibits Swarming and Virulence Factors and Increases Polymyxin B Susceptibility in Proteus mirabilis

PubMed Central

Wang, Won-Bo; Yuan, Yu-Han; Hsueh, Po-Ren; Liaw, Shwu-Jen

2012-01-01

In this study, we demonstrated that 10′(Z), 13′(E)-heptadecadienylhydroquinone (HQ17-2), isolated from the lacquer tree, could decrease swarming motility and hemolysin activity but increase polymyxin B (PB) susceptibilityof Proteus mirabilis which is intrinsically highly-resistant to PB. The increased PB susceptibility induced by HQ17-2 was also observed in clinical isolates and biofilm-grown cells. HQ17-2 could inhibit swarming in the wild-type and rppA mutant but not in the rcsB mutant, indicating that HQ17-2 inhibits swarming through the RcsB-dependent pathway, a two-component signaling pathway negatively regulating swarming and virulence factor expression. The inhibition of hemolysin activity by HQ17-2 is also mediated through the RcsB-dependent pathway, because HQ17-2 could not inhibit hemolysin activity in the rcsB mutant. Moreover, the finding that HQ17-2 inhibits the expression of flhDC gene in the wild-type and rcsB-complemented strain but not in the rcsB mutant supports the notion. By contrast, HQ17-2 could increase PB susceptibility in the wild-type and rcsB mutant but not in the rppA mutant, indicating that HQ17-2 increases PB susceptibility through the RppA-dependent pathway, a signaling pathway positively regulating PB resistance. In addition, HQ17-2 could inhibit the promoter activities of rppA and pmrI, a gene positively regulated by RppA and involved in PB resistance, in the wild-type but not in the rppA mutant. The inhibition of rppA and pmrI expression caused lipopolysaccharide purified from HQ17-2-treated cells to have higher affinity for PB. Altogether, this study uncovers new biological effects of HQ17-2 and provides evidence for the potential of HQ17-2 in clinical applications. PMID:23029100

Lambda-cyhalothrin disrupts the up-regulation effect of 17β-estradiol on post-synaptic density 95 protein expression via estrogen receptor α-dependent Akt pathway.

PubMed

Wang, Qunan; Xia, Xin; Deng, Xiaomei; Li, Nian; Wu, Daji; Zhang, Long; Yang, Chengwei; Tao, Fangbiao; Zhou, Jiangning

2016-03-01

Lambda-cyhalothrin (LCT), one of the type II pyrethroids, has been widely used throughout the world. The estrogenic effect of LCT to increase cell proliferation has been well established. However, whether the estrogenic effect of LCT will influence neurodevelopment has not been investigated. In addition, 17β-Estradiol (E2) plays a crucial role in neurodevelopment and induces an increase in synaptic proteins. The post-synaptic density 95 (PSD95) protein, which is involved in the development of the structure and function of new spines and localized with estrogen receptor α (ERα) at the post-synaptic density (PSD), was detected in our study by using hippocampal neuron cell line HT22. We found that LCT up-regulated PSD95 and ERα expression, estrogen receptor (ER) antagonist ICI182,780 and phosphatidylinositol-4; 5-bisphosphate 3-kinase (PI3K) inhibitor LY294,002 blocked this effect. In addition, LCT disrupted the promotion effect of E2 on PSD95. To investigate whether the observed changes are caused by ERα-dependent signaling activation, we next detected the effects of LCT on the ERα-mediated PI3K-Protein kinase B (PKB/Akt)-eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1) pathway. There existed an activation of Akt and the downstream factor 4E-BP1 after LCT treatment. In addition, LCT could disrupt the activation effect of E2 on the Akt pathway. However, no changes in cAMP response element-binding protein (CREB) activation and PSD95 messenger ribonucleic acid (mRNA) were observed. Our findings demonstrated that LCT could increase the PSD95 protein level via the ERα-dependent Akt pathway, and LCT might disrupt the up-regulation effect of E2 on PSD95 protein expression via this signaling pathway. Copyright © 2015. Published by Elsevier B.V.

Glutamine-dependent carbamoyl-phosphate synthetase and other enzyme activities related to the pyrimidine pathway in spleen of Squalus acanthias (spiny dogfish).

PubMed Central

Anderson, P M

1989-01-01

The first two steps of urea synthesis in liver of marine elasmobranchs involve formation of glutamine from ammonia and of carbamoyl phosphate from glutamine, catalysed by glutamine synthetase and carbamoyl-phosphate synthetase, respectively [Anderson & Casey (1984) J. Biol. Chem. 259, 456-462]; both of these enzymes are localized exclusively in the mitochondrial matrix. The objective of this study was to establish the enzymology of carbamoyl phosphate formation and utilization for pyrimidine nucleotide biosynthesis in Squalus acanthias (spiny dogfish), a representative elasmobranch. Aspartate carbamoyltransferase could not be detected in liver of dogfish. Spleen extracts, however, had glutamine-dependent carbamoyl-phosphate synthetase, aspartate carbamoyltransferase, dihydro-orotase, and glutamine synthetase activities, all localized in the cytosol; dihydro-orotate dehydrogenase, orotate phosphoribosyltransferase, and orotidine-5'-decarboxylase activities were also present. Except for glutamine synthetase, the levels of all activities were very low. The carbamoyl-phosphate synthetase activity is inhibited by UTP and is activated by 5-phosphoribosyl 1-pyrophosphate. The first three enzyme activities of the pyrimidine pathway were eluted in distinctly different positions during gel filtration chromatography under a number of different conditions; although complete proteolysis of inter-domain regions of a multifunctional complex during extraction cannot be excluded, the evidence suggests that in dogfish, in contrast to mammalian species, these three enzymes of the pyrimidine pathway exist as individual polypeptide chains. These results: (1) establish that dogfish express two different glutamine-dependent carbamoyl-phosphate synthetase activities, (2) confirm the report [Smith, Ritter & Campbell (1987) J. Biol. Chem. 262, 198-202] that dogfish express two different glutamine synthetases, and (3) provide indirect evidence that glutamine may not be available in liver for biosynthetic reactions other than urea formation. Images Fig. 1. PMID:2570570

Calcium-independent haemolysis via the lectin pathway of complement activation in the guinea-pig and other *

PubMed Central

Zhang, Y; Suankratay, C; Zhang, X-H; Jones, D R; Lint, T F; Gewurz, H

1999-01-01

We previously reported that complement-dependent haemolysis of sheep erythrocytes (E) coated with mannan (M) and sensitized with human mannan-binding lectin (MBL) via the lectin pathway in man occurs in Mg-EGTA and requires alternative pathway amplification. Calcium was required for MBL binding to E-M, but once the E-M-MBL intermediate was formed, MBL was retained and haemolysis occurred in the absence of calcium. Comparable or greater lectin pathway haemolysis in the absence of calcium was observed upon incubation of E-M-MBL in guinea-pig, rat, dog and pig sera, and was further investigated in the guinea-pig, in which titres were much higher (∼14-fold) than in man, and in contrast to humans, greater than classical pathway haemolytic activity. As in human serum, no lysis was observed in C4- or C2-deficient guinea-pig serum until purified C4 or C2, respectively, were restored. However, lectin pathway haemolytic activity in the guinea-pig did not require the alternative pathway. Removal (>98%) of factor D activity by three sequential passages through Sephadex G-75, resulting in serum which retained a normal classical pathway but no alternative pathway haemolytic activity, did not reduce the ability of guinea-pig serum to mediate haemolysis via the lectin pathway. Further, the C3-convertase formed via the lectin pathway (E-M-MBL-C4,2) lysed in C2-deficient guinea-pig but not human serum chelated with EDTA, a condition which precludes alternative pathway amplification. Thus, lectin pathway haemolysis occurs efficiently in guinea-pig serum, in the absence of calcium and without requirement for alternative pathway amplification. The guinea-pig provides a model for studying the assembly and haemolytic function of a lectin pathway which contrasts with the lectin pathway of man, and allows for comparisons that may help clarify the role of this pathway in complement biology. PMID:10457224

Inflammatory stimuli promote growth and invasion of pancreatic cancer cells through NF-κB pathway dependent repression of PP2Ac

PubMed Central

Tao, Min; Liu, Lu; Shen, Meng; Zhi, Qiaoming; Gong, Fei-Ran; Zhou, Binhua P.; Wu, Yadi; Liu, Haiyan; Chen, Kai; Shen, Bairong; Wu, Meng-Yao; Shou, Liu-Mei; Li, Wei

2016-01-01

ABSTRACT Previous studies have indicated that inflammatory stimulation represses protein phosphatase 2A (PP2A), a well-known tumor suppressor. However, whether PP2A repression participates in pancreatic cancer progression has not been verified. We used lipopolysaccharide (LPS) and macrophage-conditioned medium (MCM) to establish in vitro inflammation models, and investigated whether inflammatory stimuli affect pancreatic cancer cell growth and invasion PP2A catalytic subunit (PP2Ac)-dependently. Via nude mouse models of orthotopic tumor xenografts and dibutyltin dichloride (DBTC)-induced chronic pancreatitis, we evaluated the effect of an inflammatory microenvironment on PP2Ac expression in vivo. We cloned the PP2Acα and PP2Acβ isoform promoters to investigate the PP2Ac transcriptional regulation mechanisms. MCM accelerated pancreatic cancer cell growth; MCM and LPS promoted cell invasion. DBTC promoted xenograft growth and metastasis, induced tumor-associated macrophage infiltration, promoted angiogenesis, activated the nuclear factor-κB (NF-κB) pathway, and repressed PP2Ac expression. In vitro, LPS and MCM downregulated PP2Ac mRNA and protein. PP2Acα overexpression attenuated JNK, ERK, PKC, and IKK phosphorylation, and impaired LPS/MCM-stimulated cell invasion and MCM-promoted cell growth. LPS and MCM activated the NF-κB pathway in vitro. LPS and MCM induced IKK and IκB phosphorylation, leading to p65/RelA nuclear translocation and transcriptional activation. Overexpression of the dominant negative forms of IKKα attenuated LPS and MCM downregulation of PP2Ac, suggesting inflammatory stimuli repress PP2Ac expression NF-κB pathway–dependently. Luciferase reporter gene assay verified that LPS and MCM downregulated PP2Ac transcription through an NF-κB–dependent pathway. Our study presents a new mechanism in inflammation-driven cancer progression through NF-κB pathway–dependent PP2Ac repression. PMID:26761431

Aldosterone does not require angiotensin II to activate NCC through a WNK4-SPAK-dependent pathway.

PubMed

van der Lubbe, Nils; Lim, Christina H; Meima, Marcel E; van Veghel, Richard; Rosenbaek, Lena Lindtoft; Mutig, Kerim; Danser, Alexander H J; Fenton, Robert A; Zietse, Robert; Hoorn, Ewout J

2012-06-01

We and others have recently shown that angiotensin II can activate the sodium chloride cotransporter (NCC) through a WNK4-SPAK-dependent pathway. Because WNK4 was previously shown to be a negative regulator of NCC, it has been postulated that angiotensin II converts WNK4 to a positive regulator. Here, we ask whether aldosterone requires angiotensin II to activate NCC and if their effects are additive. To do so, we infused vehicle or aldosterone in adrenalectomized rats that also received the angiotensin receptor blocker losartan. In the presence of losartan, aldosterone was still capable of increasing total and phosphorylated NCC twofold to threefold. The kinases WNK4 and SPAK also increased with aldosterone and losartan. A dose-dependent relationship between aldosterone and NCC, SPAK, and WNK4 was identified, suggesting that these are aldosterone-sensitive proteins. As more functional evidence of increased NCC activity, we showed that rats receiving aldosterone and losartan had a significantly greater natriuretic response to hydrochlorothiazide than rats receiving losartan only. To study whether angiotensin II could have an additive effect, rats receiving aldosterone with losartan were compared with rats receiving aldosterone only. Rats receiving aldosterone only retained more sodium and had twofold to fourfold increase in phosphorylated NCC. Together, our results demonstrate that aldosterone does not require angiotensin II to activate NCC and that WNK4 appears to act as a positive regulator in this pathway. The additive effect of angiotensin II may favor electroneutral sodium reabsorption during hypovolemia and may contribute to hypertension in diseases with an activated renin-angiotensin-aldosterone system.

Membrane and Integrative Nuclear Fibroblastic Growth Factor Receptor (FGFR) Regulation of FGF-23*

PubMed Central

Han, Xiaobin; Xiao, Zhousheng; Quarles, L. Darryl

2015-01-01

Fibroblastic growth factor receptor 1 (FGFR1) signaling pathways are implicated in the regulation of FGF-23 gene transcription, but the molecular pathways remain poorly defined. We used low molecular weight (LMW, 18 kDa) FGF-2 and high molecular weight (HMW) FGF-2 isoforms, which, respectively, activate cell surface FGF receptors and intranuclear FGFR1, to determine the roles of membrane FGFRs and integrative nuclear FGFR1 signaling (INFS) in the regulation of FGF-23 gene transcription in osteoblasts. We found that LMW-FGF-2 induced NFAT and Ets1 binding to conserved cis-elements in the proximal FGF-23 promoter and stimulated FGF-23 promoter activity through PLCγ/calcineurin/NFAT and MAPK pathways in SaOS-2 and MC3T3-E1 osteoblasts. In contrast, HMW-FGF-2 stimulated FGF-23 promoter activity in osteoblasts through a cAMP-dependent binding of FGFR1 and cAMP-response element-binding protein (CREB) to a conserved cAMP response element (CRE) contiguous with the NFAT binding site in the FGF-23 promoter. Mutagenesis of the NFAT and CRE binding sites, respectively, inhibited the effects of LMW-FGF-2 and HMW-FGF-23 to stimulate FGF-23 promoter activity. FGF-2 activation of both membrane FGFRs and INFS-dependent FGFR1 pathways may provide a means to integrate systemic and local regulation of FGF-23 transcription under diverse physiological and pathological conditions. PMID:25752607

Reporter Gene-Facilitated Detection of Compounds in Arabidopsis Leaf Extracts that Activate the Karrikin Signaling Pathway.

PubMed

Sun, Yueming K; Flematti, Gavin R; Smith, Steven M; Waters, Mark T

2016-01-01

Karrikins are potent germination stimulants generated by the combustion of plant matter. Treatment of Arabidopsis with karrikins triggers a signaling process that is dependent upon a putative receptor protein KARRIKIN INSENSITIVE 2 (KAI2). KAI2 is a homolog of DWARF 14 (D14), the receptor for endogenous strigolactone hormones. Genetic analyses suggest that KAI2 also perceives endogenous signal(s) that are not strigolactones. Activation of KAI2 by addition of karrikins to Arabidopsis plants induces expression of transcripts including D14-LIKE 2 ( DLK2 ). We constructed the synthetic reporter gene DLK2 : LUC in Arabidopsis , which comprises the firefly luciferase gene ( LUC ) driven by the DLK2 promoter. Here we describe a luminescence-based reporter assay with Arabidopsis seeds to detect chemical signals that can activate the KAI2 signaling pathway. We demonstrate that the DLK2 : LUC assay can selectively and sensitively detect karrikins and a functionally similar synthetic strigolactone analog. Crucially we show that crude extracts from Arabidopsis leaves can also activate DLK2 : LUC in a KAI2-dependent manner. Our work provides the first direct evidence for the existence of endogenous chemical signals that can activate the KAI2-mediated signaling pathway in Arabidopsis . This sensitive reporter system can now be used for the bioassay-guided purification and identification of putative endogenous KAI2 ligands or their precursors, and endogenous compounds that might modulate the KAI2 signaling pathway.

Reporter Gene-Facilitated Detection of Compounds in Arabidopsis Leaf Extracts that Activate the Karrikin Signaling Pathway

PubMed Central

Sun, Yueming K.; Flematti, Gavin R.; Smith, Steven M.; Waters, Mark T.

2016-01-01

Karrikins are potent germination stimulants generated by the combustion of plant matter. Treatment of Arabidopsis with karrikins triggers a signaling process that is dependent upon a putative receptor protein KARRIKIN INSENSITIVE 2 (KAI2). KAI2 is a homolog of DWARF 14 (D14), the receptor for endogenous strigolactone hormones. Genetic analyses suggest that KAI2 also perceives endogenous signal(s) that are not strigolactones. Activation of KAI2 by addition of karrikins to Arabidopsis plants induces expression of transcripts including D14-LIKE 2 (DLK2). We constructed the synthetic reporter gene DLK2:LUC in Arabidopsis, which comprises the firefly luciferase gene (LUC) driven by the DLK2 promoter. Here we describe a luminescence-based reporter assay with Arabidopsis seeds to detect chemical signals that can activate the KAI2 signaling pathway. We demonstrate that the DLK2:LUC assay can selectively and sensitively detect karrikins and a functionally similar synthetic strigolactone analog. Crucially we show that crude extracts from Arabidopsis leaves can also activate DLK2:LUC in a KAI2-dependent manner. Our work provides the first direct evidence for the existence of endogenous chemical signals that can activate the KAI2-mediated signaling pathway in Arabidopsis. This sensitive reporter system can now be used for the bioassay-guided purification and identification of putative endogenous KAI2 ligands or their precursors, and endogenous compounds that might modulate the KAI2 signaling pathway. PMID:27994609

Basic Fibroblast Growth Factor Activates Serum Response Factor Gene Expression by Multiple Distinct Signaling Mechanisms

PubMed Central

Spencer, Jeffrey A.; Major, Michael L.; Misra, Ravi P.

1999-01-01

Serum response factor (SRF) plays a central role in the transcriptional response of mammalian cells to a variety of extracellular signals. It is a key regulator of many cellular early response genes which are believed to be involved in cell growth and differentiation. The mechanism by which SRF activates transcription in response to mitogenic agents has been extensively studied; however, significantly less is known about regulation of the SRF gene itself. Previously, we identified distinct regulatory elements in the SRF promoter that play a role in activation, including a consensus ETS domain binding site, a consensus overlapping Sp/Egr-1 binding site, and two SRF binding sites. We further showed that serum induces SRF by a mechanism that requires an intact SRF binding site, also termed a CArG box. In the present study we demonstrate that in response to stimulation of cells by a purified growth factor, basic fibroblast growth factor (bFGF), the SRF promoter is upregulated by a complex pathway that involves at least two independent mechanisms: a CArG box-independent mechanism that is mediated by an ETS binding site, and a novel CArG box-dependent mechanism that requires both an Sp factor binding site and the CArG motifs for maximal stimulation. Our analysis indicates that the CArG/Sp element activation mechanism is mediated by distinct signaling pathways. The CArG box-dependent component is targeted by a Rho-mediated pathway, and the Sp binding site-dependent component is targeted by a Ras-mediated pathway. Both SRF and bFGF have been implicated in playing an important role in mediating cardiogenesis during development. The implications of our findings for SRF expression during development are discussed. PMID:10330138

PEBP1, a RAF kinase inhibitory protein, negatively regulates starvation-induced autophagy by direct interaction with LC3.

PubMed

Noh, Hae Sook; Hah, Young-Sool; Zada, Sahib; Ha, Ji Hye; Sim, Gyujin; Hwang, Jin Seok; Lai, Trang Huyen; Nguyen, Huynh Quoc; Park, Jae-Yong; Kim, Hyun Joon; Byun, June-Ho; Hahm, Jong Ryeal; Kang, Kee Ryeon; Kim, Deok Ryong

2016-11-01

Autophagy plays a critical role in maintaining cell homeostasis in response to various stressors through protein conjugation and activation of lysosome-dependent degradation. MAP1LC3B/LC3B (microtubule- associated protein 1 light chain 3 β) is conjugated with phosphatidylethanolamine (PE) in the membranes and regulates initiation of autophagy through interaction with many autophagy-related proteins possessing an LC3-interacting region (LIR) motif, which is composed of 2 hydrophobic amino acids (tryptophan and leucine) separated by 2 non-conserved amino acids (WXXL). In this study, we identified a new putative LIR motif in PEBP1/RKIP (phosphatidylethanolamine binding protein 1) that was originally isolated as a PE-binding protein and also a cellular inhibitor of MAPK/ERK signaling. PEBP1 was specifically bound to PE-unconjugated LC3 in cells, and mutation (WXXL mutated to AXXA) of this LIR motif disrupted its interaction with LC3 proteins. Interestingly, overexpression of PEBP1 significantly inhibited starvation-induced autophagy by activating the AKT and MTORC1 (mechanistic target of rapamycin [serine/threonine kinase] complex 1) signaling pathway and consequently suppressing the ULK1 (unc-51 like autophagy activating kinase 1) activity. In contrast, ablation of PEBP1 expression dramatically promoted the autophagic process under starvation conditions. Furthermore, PEBP1 lacking the LIR motif highly stimulated starvation-induced autophagy through the AKT-MTORC1-dependent pathway. PEBP1 phosphorylation at Ser153 caused dissociation of LC3 from the PEBP1-LC3 complex for autophagy induction. PEBP1-dependent suppression of autophagy was not associated with the MAPK pathway. These findings suggest that PEBP1 can act as a negative mediator in autophagy through stimulation of the AKT-MTORC1 pathway and direct interaction with LC3.

Honokiol and magnolol stimulate glucose uptake by activating PI3K-dependent Akt in L6 myotubes.

PubMed

Choi, Sun-Sil; Cha, Byung-Yoon; Lee, Young-Sil; Yonezawa, Takayuki; Teruya, Toshiaki; Nagai, Kazuo; Woo, Je-Tae

2012-01-01

Honokiol and magnolol, ingredients of Magnolia officinalis, which is used in traditional Chinese and Japanese medicines, have been reported to have antioxidant, anticancer, and antiangiogenic effects. Effects of these compounds on glucose metabolism in adipocytes have also been reported. However, their effects on skeletal muscle glucose uptake and the underlying molecular mechanisms are still unknown. Here, we investigated the direct effects and signaling pathways activated by honokiol and magnolol in skeletal muscle cells using L6 myotubes. We found that honokiol and magnolol dose-dependently acutely stimulated glucose uptake without synergistic effects of combined administration in L6 myotubes. Treatment with honokiol and magnolol also stimulated glucose transporter-4 translocation to the cell surface. Honokiol- and magnolol-stimulated glucose uptake was blocked by the phosphatidylinositol-3 kinase inhibitor, wortmannin. Both honokiol and magnolol stimulated Akt phosphorylation, a key element in the insulin signaling pathway, which was completely inhibited by wortmannin. These results suggest that honokiol and magnolol might have beneficial effects on glucose metabolism by activating the insulin signaling pathway. Copyright © 2012 International Union of Biochemistry and Molecular Biology, Inc.

Sinularin Induces Apoptosis through Mitochondria Dysfunction and Inactivation of the pI3K/Akt/mTOR Pathway in Gastric Carcinoma Cells.

PubMed

Wu, Yu-Jen; Wong, Bing-Sang; Yea, Shu-Hao; Lu, Chi-I; Weng, Shun-Hsiang

2016-07-27

Sinularin is an active compound isolated from the cultured soft coral Sinularia flexibilis. In this study, we investigated the effects of sinularin on two human gastric cancer cell lines, AGS and NCI-N87. Our results demonstrated that sinularin suppressed the proliferation of gastric cancer cells in a dose-dependent manner and induced apoptosis. In addition, the loss of mitochondrial membrane potential, the release of cytochrome C, the activation of Bax, Bad and caspase-3/9, and the suppression of p-Bad, Bcl-xL and Bcl-2 were observed in the cells treated with sinularin. This finding suggests that sinularin-induced apoptosis is associated with mitochondria-mediated apoptosis and occurs through caspase-dependent pathways. Furthermore, sinularin inhibited the phosphoinositol 3-kinase/Akt/mechanistic target of the rapamycin signaling pathway. Taken together, our results show that sinularin-induced apoptosis is mediated by activation of the caspase cascade and mitochondrial dysfunction. Our findings suggest that sinularin merits further evaluation as a chemotherapeutic agent for human gastric cancer.

Optical control of neuronal activity using a light-operated GIRK channel opener (LOGO).

PubMed

Barber, David M; Schönberger, Matthias; Burgstaller, Jessica; Levitz, Joshua; Weaver, C David; Isacoff, Ehud Y; Baier, Herwig; Trauner, Dirk

2016-01-01

G-protein coupled inwardly rectifying potassium channels (GIRKs) are ubiquitously expressed throughout the human body and are an integral part of inhibitory signal transduction pathways. Upon binding of G βγ subunits released from G-protein coupled receptors (GPCRs), GIRK channels open and reduce the activity of excitable cells via hyperpolarization. As such, they play a role in cardiac output, the coordination of movement and cognition. Due to their involvement in a multitude of pathways, the precision control of GIRK channels is an important endeavour. Here, we describe the development of the photoswitchable agonist LOGO (the L ight O perated G IRK-channel O pener), which activates GIRK channels in the dark and is rapidly deactivated upon exposure to long wavelength UV irradiation. LOGO is the first K + channel opener and selectively targets channels that contain the GIRK1 subunit. It can be used to optically silence action potential firing in dissociated hippocampal neurons and LOGO exhibits activity in vivo , controlling the motility of zebrafish larvae in a light dependent fashion. We envisage that LOGO will be a valuable research tool to dissect the function of GIRK channels from other GPCR dependent signalling pathways.

Effects of hepatitis C virus core protein and nonstructural protein 4B on the Wnt/β-catenin pathway.

PubMed

Jiang, Xiao-Hua; Xie, Yu-Tao; Cai, Ya-Ping; Ren, Jing; Ma, Tao

2017-05-25

Hepatitis C virus (HCV) core protein and nonstructural protein 4B (NS4B) are potentially oncogenic. Aberrant activation of the Wnt/β-catenin signaling pathway is closely associated with hepatocarcinogenesis. We investigated the effects of HCV type 1b core protein and NS4B on Wnt/β-catenin signaling in various liver cells, and explored the molecular mechanism underlying HCV-related hepatocarcinogenesis. Compared with the empty vector control, HCV core protein and NS4B demonstrated the following characteristics in the Huh7 cells: significantly enhanced β-catenin/Tcf-dependent transcriptional activity (F = 40.87, P < 0.01); increased nuclear translocation of β-catenin (F = 165.26, P < 0.01); upregulated nuclear β-catenin, cytoplasmic β-catenin, Wnt1, c-myc, and cyclin D1 protein expression (P < 0.01); and promoted proliferation of Huh7 cells (P < 0.01 or P < 0.05). Neither protein enhanced β-catenin/Tcf-dependent transcriptional activity in the LO2 cells (F = 0.65, P > 0.05), but they did significantly enhance Wnt3a-induced β-catenin/Tcf-dependent transcriptional activity (F = 64.25, P < 0.01), and promoted the nuclear translocation of β-catenin (F = 66.54, P < 0.01) and the Wnt3a-induced proliferation of LO2 cells (P < 0.01 or P < 0.05). Moreover, activation of the Wnt/β-catenin signaling pathway was greater with the core protein than with NS4B (P < 0.01 or P < 0.05). HCV core protein and NS4B directly activate the Wnt/β-catenin signaling pathway in Huh7 cells and LO2 cells induced by Wnt3a. These data suggest that HCV core protein and NS4B contribute to HCV-associated hepatocellular carcinogenesis.

Molecular evolution of multiple-level control of heme biosynthesis pathway in animal kingdom.

PubMed

Tzou, Wen-Shyong; Chu, Ying; Lin, Tzung-Yi; Hu, Chin-Hwa; Pai, Tun-Wen; Liu, Hsin-Fu; Lin, Han-Jia; Cases, Ildeofonso; Rojas, Ana; Sanchez, Mayka; You, Zong-Ye; Hsu, Ming-Wei

2014-01-01

Adaptation of enzymes in a metabolic pathway can occur not only through changes in amino acid sequences but also through variations in transcriptional activation, mRNA splicing and mRNA translation. The heme biosynthesis pathway, a linear pathway comprised of eight consecutive enzymes in animals, provides researchers with ample information for multiple types of evolutionary analyses performed with respect to the position of each enzyme in the pathway. Through bioinformatics analysis, we found that the protein-coding sequences of all enzymes in this pathway are under strong purifying selection, from cnidarians to mammals. However, loose evolutionary constraints are observed for enzymes in which self-catalysis occurs. Through comparative genomics, we found that in animals, the first intron of the enzyme-encoding genes has been co-opted for transcriptional activation of the genes in this pathway. Organisms sense the cellular content of iron, and through iron-responsive elements in the 5' untranslated regions of mRNAs and the intron-exon boundary regions of pathway genes, translational inhibition and exon choice in enzymes may be enabled, respectively. Pathway product (heme)-mediated negative feedback control can affect the transport of pathway enzymes into the mitochondria as well as the ubiquitin-mediated stability of enzymes. Remarkably, the positions of these controls on pathway activity are not ubiquitous but are biased towards the enzymes in the upstream portion of the pathway. We revealed that multiple-level controls on the activity of the heme biosynthesis pathway depend on the linear depth of the enzymes in the pathway, indicating a new strategy for discovering the molecular constraints that shape the evolution of a metabolic pathway.

Physical activity and nicotine dependence among a national sample of young U.S. adults who smoke daily: evaluation of cross-sectional and longitudinal associations to determine which behavior drives this relationship.

PubMed

Loprinzi, Paul D; Kane, Christy J; Mahoney, Sara; Walker, Jerome F

2015-02-01

The association between nicotine dependence and physical activity (PA) is relatively unknown. No study has concurrently examined the cross-sectional and longitudinal associations between PA and nicotine dependence, which was the primary purpose of this study. A secondary purpose was to examine how well nicotine dependence and PA behavior track over a two-year period. Data from the 2003-2005 National Youth Smoking Cessation Survey (NYSCS) were used, with young adults (18-24 yrs; n=1168) being followed over a two-year period. Physical activity was assessed using a questionnaire and nicotine dependence was assessed using the modified Fagerstrom Test for Nicotine Dependence scale. This study identified three notable findings: 1) baseline PA and nicotine dependence demonstrated a bidirectional, cross-sectional association (e.g., β=-0.23; 95% CI: -0.44 to -0.02; p=0.02); 2) when examined longitudinally, nicotine dependence influenced PA (OR=0.90; 95% CI: 0.82-0.99; p=0.04), but there was no evidence of the reverse pathway (i.e., PA influencing 2-year follow-up smoking status [OR=0.95; 95% CI: 0.66-1.39; p=0.82) or nicotine dependence (β=0.05; 95% CI: -0.14 to 0.24, p=0.61]); and 3) both PA (OR=3.52, 95% CI: 2.68-4.69; p<0.001) and nicotine dependence (β=0.52; 95% CI: 0.46-0.58, p<0.001) tracked relatively well over a two-year period during early adulthood. These findings suggest that both behaviors (physical activity and nicotine dependence) track over time, but nicotine dependence appears to be driving the cross-sectional relationship between nicotine dependence and physical activity, as opposed to the reverse pathway. Copyright © 2014 Elsevier Inc. All rights reserved.

Ubiquitylation and the Fanconi Anemia Pathway

PubMed Central

Garner, Elizabeth; Smogorzewska, Agata

2012-01-01

The Fanconi anemia (FA) pathway maintains genome stability through co-ordination of DNA repair of interstrand crosslinks (ICLs). Disruption of the FA pathway yields hypersensitivity to interstrand crosslinking agents, bone marrow failure and cancer predisposition. Early steps in DNA damage dependent activation of the pathway are governed by monoubiquitylation of FANCD2 and FANCI by the intrinsic FA E3 ubiquitin ligase, FANCL. Downstream FA pathway components and associated factors such as FAN1 and SLX4 exhibit ubiquitin-binding motifs that are important for their DNA repair function, underscoring the importance of ubiquitylation in FA pathway mediated repair. Importantly, ubiquitylation provides the foundations for cross-talk between repair pathways, which in concert with the FA pathway, resolve interstrand crosslink damage and maintain genomic stability. PMID:21605559

Bcl-2 Blocks a Caspase-Dependent Pathway of Apoptosis Activated by Herpes Simplex Virus 1 Infection in HEp-2 Cells

PubMed Central

Galvan, Veronica; Brandimarti, Renato; Munger, Joshua; Roizman, Bernard

2000-01-01

Earlier reports have shown that herpes simplex virus 1 (HSV-1) mutants induce programmed cell death and that wild-type virus blocks the execution of the cell death program triggered by expression of viral genes, by the Fas and tumor necrosis factor pathways, or by nonspecific stress agents. In particular, an earlier report from this laboratory showed that the mutant virus d120 lacking the genes encoding infected cell protein 4 (ICP4), the major regulatory protein of the virus, induces a caspase-3-independent pathway of apoptosis in human SK-N-SH cells. Here we report that the pathway of apoptosis induced by the d120 mutant in human HEp-2 cells is caspase dependent. Specifically, in HEp-2 cells infected with d120, (i) a broad-range inhibitor of caspase activity, z-vad-FMK, efficiently blocked DNA fragmentation, (ii) cytochrome c was released into the cytoplasm, (iii) caspase-3 was activated inasmuch as poly(ADP-ribose) polymerase was cleaved, and (iv) chromatin condensation and fragmentation of cellular DNA were observed. In parallel studies, HEp-2 cells were transfected with a plasmid encoding human Bcl-2 and a clone (VAX-3) expressing high levels of Bcl-2 was selected. This report shows that Bcl-2 blocked all of the manifestations associated with programmed cell death caused by infection with the d120 mutant. Consistent with their resistance to programmed cell death, VAX-3 cells overproduced infected cell protein 0 (ICP0). An unexpected observation was that ICP0 encoded by the d120 mutant accumulated late in infection in small, quasi-uniform vesicle-like structures in all cell lines tested. Immunofluorescence-based colocalization studies indicated that these structures were not mitochondria or components of the endoplasmic reticulum or the late endosomal compartment. These studies affirm the conclusion that HSV can induce programmed cell death at multiple steps in the course of its replication, that the d120 mutant can induce both caspase-dependent and -independent pathways of programmed cell death, and that virus-induced stimuli of programmed cell death may differ with respect to the pathway that they activate. PMID:10644366

Redox regulation of cardiomyocyte cell cycling via an ERK1/2 and c-Myc-dependent activation of cyclin D2 transcription

PubMed Central

Murray, Thomas V.A.; Smyrnias, Ioannis; Schnelle, Moritz; Mistry, Rajesh K.; Zhang, Min; Beretta, Matteo; Martin, Daniel; Anilkumar, Narayana; de Silva, Shana M.; Shah, Ajay M.; Brewer, Alison C.

2015-01-01

Adult mammalian cardiomyocytes have a very limited capacity to proliferate, and consequently the loss of cells after cardiac stress promotes heart failure. Recent evidence suggests that administration of hydrogen peroxide (H2O2), can regulate redox-dependent signalling pathway(s) to promote cardiomyocyte proliferation in vitro, but the potential relevance of such a pathway in vivo has not been tested. We have generated a transgenic (Tg) mouse model in which the H2O2-generating enzyme, NADPH oxidase 4 (Nox4), is overexpressed within the postnatal cardiomyocytes, and observed that the hearts of 1–3 week old Tg mice pups are larger in comparison to wild type (Wt) littermate controls. We demonstrate that the cardiomyocytes of Tg mouse pups have increased cell cycling capacity in vivo as determined by incorporation of 5-bromo-2′-deoxyuridine. Further, microarray analyses of the transcriptome of these Tg mouse hearts suggested that the expression of cyclin D2 is significantly increased. We investigated the molecular mechanisms which underlie this more proliferative phenotype in isolated neonatal rat cardiomyocytes (NRCs) in vitro, and demonstrate that Nox4 overexpression mediates an H2O2-dependent activation of the ERK1/2 signalling pathway, which in turn phosphorylates and activates the transcription factor c-myc. This results in a significant increase in cyclin D2 expression, which we show to be mediated, at least in part, by cis-acting c-myc binding sites within the proximal cyclin D2 promoter. Overexpression of Nox4 in NRCs results in an increase in their proliferative capacity that is ablated by the silencing of cyclin D2. We further demonstrate activation of the ERK1/2 signalling pathway, increased phosphorylation of c-myc and significantly increased expression of cyclin D2 protein in the Nox4 Tg hearts. We suggest that this pathway acts to maintain the proliferative capacity of cardiomyocytes in Nox4 Tg pups in vivo and so delays their exit from the cell cycle after birth. PMID:25450615

Prx I Suppresses K-ras-Driven Lung Tumorigenesis by Opposing Redox-Sensitive ERK/Cyclin D1 Pathway

PubMed Central

Park, Young-Ho; Kim, Sun-Uk; Lee, Bo-Kyoung; Kim, Hyun-Sun; Song, In-Sung; Shin, Hye-Jun; Han, Ying-Hao; Chang, Kyu-Tae; Kim, Jin-Man; Lee, Dong-Seok; Kim, Yeul-Hong; Choi, Chang-Min; Kim, Bo-Yeon

2013-01-01

Abstract Aims: Coupled responses of mutated K-ras and oxidative stress are often an important etiological factor in non–small-cell lung cancer (NSCLC). However, relatively few studies have examined the control mechanism of oxidative stress in oncogenic K-ras-driven NSCLC progression. Here, we studied whether the redox signaling pathway governed by peroxiredoxin I (Prx I) is involved in K-rasG12D-mediated lung adenocarcinogenesis. Results: Using human-lung adenocarcinoma tissues and lung-specific K-rasG12D-transgenic mice, we found that Prx I was significantly up-regulated in the tumor regions via activation of nuclear erythroid 2-related factor 2 (Nrf2) transcription. Interestingly, the increased reactive oxygen species (ROS) by null mutation of Prx I greatly promoted K-rasG12D-driven lung tumorigenesis in number and size, which appeared to require the activation of the ROS-dependent extracellular signal-regulated kinase (ERK)/cyclin D1 pathway. Innovation: Taken together, these results suggest that Prx I functions as an Nrf2-dependently inducible tumor suppressant in K-ras-driven lung adenocarcinogenesis by opposing ROS/ERK/cyclin D1 pathway activation. Conclusion: These findings provide a better understanding of oxidative stress-mediated lung tumorigenesis. Antioxid. Redox Signal. 19, 482–496. PMID:23186333

Loss of Pancreas upon Activated Wnt Signaling Is Concomitant with Emergence of Gastrointestinal Identity

PubMed Central

Herrero-Martin, Griselda; Puri, Sapna; Taketo, Makoto Mark; Rojas, Anabel; Hebrok, Matthias; Cano, David A.

2016-01-01

Organ formation is achieved through the complex interplay between signaling pathways and transcriptional cascades. The canonical Wnt signaling pathway plays multiple roles during embryonic development including patterning, proliferation and differentiation in distinct tissues. Previous studies have established the importance of this pathway at multiple stages of pancreas formation as well as in postnatal organ function and homeostasis. In mice, gain-of-function experiments have demonstrated that activation of the canonical Wnt pathway results in pancreatic hypoplasia, a phenomenon whose underlying mechanisms remains to be elucidated. Here, we show that ectopic activation of epithelial canonical Wnt signaling causes aberrant induction of gastric and intestinal markers both in the pancreatic epithelium and mesenchyme, leading to the development of gut-like features. Furthermore, we provide evidence that β -catenin-induced impairment of pancreas formation depends on Hedgehog signaling. Together, our data emphasize the developmental plasticity of pancreatic progenitors and further underscore the key role of precise regulation of signaling pathways to maintain appropriate organ boundaries. PMID:27736991

Activation of DNA Damage Repair Pathways by Murine Polyomavirus

PubMed Central

Heiser, Katie; Nicholas, Catherine; Garcea, Robert L.

2016-01-01

Nuclear replication of DNA viruses activates DNA damage repair (DDR) pathways, which are thought to detect and inhibit viral replication. However, many DNA viruses also depend on these pathways in order to optimally replicate their genomes. We investigated the relationship between murine polyomavirus (MuPyV) and components of DDR signaling pathways including CHK1, CHK2, H2AX, ATR, and DNAPK. We found that recruitment and retention of DDR proteins at viral replication centers was independent of H2AX, as well as the viral small and middle T-antigens. Additionally, infectious virus production required ATR kinase activity, but was independent of CHK1, CHK2, or DNAPK signaling. ATR inhibition did not reduce the total amount of viral DNA accumulated, but affected the amount of virus produced, indicating a defect in virus assembly. These results suggest that MuPyV may utilize a subset of DDR proteins or non-canonical DDR signaling pathways in order to efficiently replicate and assemble. PMID:27529739

Aldolase positively regulates of the canonical Wnt signaling pathway

PubMed Central

2014-01-01

The Wnt signaling pathway is an evolutionary conserved system, having pivotal roles during animal development. When over-activated, this signaling pathway is involved in cancer initiation and progression. The canonical Wnt pathway regulates the stability of β-catenin primarily by a destruction complex containing a number of different proteins, including Glycogen synthase kinase 3β (GSK-3β) and Axin, that promote proteasomal degradation of β-catenin. As this signaling cascade is modified by various proteins, novel screens aimed at identifying new Wnt signaling regulators were conducted in our laboratory. One of the different genes that were identified as Wnt signaling activators was Aldolase C (ALDOC). Here we report that ALDOC, Aldolase A (ALDOA) and Aldolase B (ALDOB) activate Wnt signaling in a GSK-3β-dependent mechanism, by disrupting the GSK-3β-Axin interaction and targeting Axin to the dishevelled (Dvl)-induced signalosomes that positively regulate the Wnt pathway thus placing the Aldolase proteins as novel Wnt signaling regulators. PMID:24993527

Cutting edge: A transcriptional repressor and corepressor induced by the STAT3-regulated anti-inflammatory signaling pathway.

PubMed

El Kasmi, Karim C; Smith, Amber M; Williams, Lynn; Neale, Geoffrey; Panopoulos, Athanasia D; Panopolous, Athanasia; Watowich, Stephanie S; Häcker, Hans; Foxwell, Brian M J; Murray, Peter J

2007-12-01

IL-10 regulates anti-inflammatory signaling via the activation of STAT3, which in turn controls the induction of a gene expression program whose products execute inhibitory effects on proinflammatory mediator production. In this study we show that IL-10 induces the expression of an ETS family transcriptional repressor, ETV3, and a helicase family corepressor, Strawberry notch homologue 2 (SBNO2), in mouse and human macrophages. IL-10-mediated induction of ETV3 and SBNO2 expression was dependent upon both STAT3 and a stimulus through the TLR pathway. We also observed that ETV3 expression was strongly induced by the STAT3 pathway regulated by IL-10 but not by STAT3 signaling activated by IL-6, which cannot activate the anti-inflammatory signaling pathway. ETV3 and SBNO2 repressed NF-kappaB- but not IFN regulatory factor 7 (IRF7)-activated transcriptional reporters. Collectively our data suggest that ETV3 and SBNO2 are components of the pathways that contribute to the downstream anti-inflammatory effects of IL-10.

Replication Protein A (RPA) deficiency activates the Fanconi anemia DNA repair pathway.

PubMed

Jang, Seok-Won; Jung, Jin Ki; Kim, Jung Min

2016-09-01

The Fanconi anemia (FA) pathway regulates DNA inter-strand crosslink (ICL) repair. Despite our greater understanding of the role of FA in ICL repair, its function in the preventing spontaneous genome instability is not well understood. Here, we show that depletion of replication protein A (RPA) activates the FA pathway. RPA1 deficiency increases chromatin recruitment of FA core complex, leading to FANCD2 monoubiquitination (FANCD2-Ub) and foci formation in the absence of DNA damaging agents. Importantly, ATR depletion, but not ATM, abolished RPA1 depletion-induced FANCD2-Ub, suggesting that ATR activation mediated FANCD2-Ub. Interestingly, we found that depletion of hSSB1/2-INTS3, a single-stranded DNA-binding protein complex, induces FANCD2-Ub, like RPA1 depletion. More interestingly, depletion of either RPA1 or INTS3 caused increased accumulation of DNA damage in FA pathway deficient cell lines. Taken together, these results indicate that RPA deficiency induces activation of the FA pathway in an ATR-dependent manner, which may play a role in the genome maintenance.

Homocysteine enhances MMP-9 production in murine macrophages via ERK and Akt signaling pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Seung Jin; Lee, Yi Sle; Seo, Kyo Won

2012-04-01

Homocysteine (Hcy) at elevated levels is an independent risk factor of cardiovascular diseases, including atherosclerosis. In the present study, we investigated the effect of Hcy on the production of matrix metalloproteinases (MMP) in murine macrophages. Among the MMP known to regulate the activities of collagenase and gelatinase, Hcy exclusively increased the gelatinolytic activity of MMP-9 in J774A.1 cells as well as in mouse peritoneal macrophages. Furthermore, this activity was found to be correlated with Western blot findings in J774A.1 cells, which showed that MMP-9 expression was concentration- and time-dependently increased by Hcy. Inhibition of the ERK and Akt pathways ledmore » to a significant decrease in Hcy-induced MMP-9 expression, and combined treatment with inhibitors of the ERK and Akt pathways showed an additive effects. Activity assays for ERK and Akt showed that Hcy increased the phosphorylation of both, but these phosphorylation were not affected by inhibitors of the Akt and ERK pathways. In line with these findings, the molecular inhibition of ERK and Akt using siRNA did not affect the Hcy-induced phosphorylation of Akt and ERK, respectively. Taken together, these findings suggest that Hcy enhances MMP-9 production in murine macrophages by separately activating the ERK and Akt signaling pathways. -- Highlights: ► Homocysteine (Hcy) induced MMP-9 production in murine macrophages. ► Hcy induced MMP-9 production through ERK and Akt signaling pathways. ► ERK and Akt signaling pathways were activated by Hcy in murine macrophages. ► ERK and Akt pathways were additively act on Hcy-induced MMP-9 production. ► Hcy enhances MMP-9 production in macrophages via activation of ERK and Akt signaling pathways in an independent manner.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Jeong Su; Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752; Kang, Dong Hoon

p70 ribosomal S6 kinase 1 (S6K1) is an important serine/threonine kinase and downstream target of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway. PF-4708671 is a specific inhibitor of S6K1, and prevents S6K1-mediated phosphorylation of the S6 protein. PF-4708671 treatment often leads to apoptotic cell death. However, the protective mechanism against PF-4708671-induced cell death has not been elucidated. The nuclear factor erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway is essential for protecting cells against oxidative stress. p62, an adaptor protein in the autophagic process, enhances Nrf2 activation through the impairment of Keap1 activity. In thismore » study, we showed that PF-4708671 induces autophagic Keap1 degradation-mediated Nrf2 activation in p62-dependent manner. Furthermore, p62-dependent Nrf2 activation plays a crucial role in protecting cells from PF-4708671-mediated apoptosis. - Highlights: • PF-4708671, a S6K1-specific inhibitor, prevents S6K1-mediated S6 phosphorylation. • However, PF-4708671 treatment often leads to apoptotic cell death. • Protective mechanism against PF-4708671-induced cell death remains to be elucidated. • PF-4708671 induced p62-dependent, autophagic Keap1 degradation-mediated Nrf2 activation. • p62-dependent Nrf2 activation protects cells from PF-4708671-mediated apoptosis.« less

Activation of innate antiviral immune response via double-stranded RNA-dependent RLR receptor-mediated necroptosis

PubMed Central

Wang, Wei; Wang, Wei-Hua; Azadzoi, Kazem M.; Su, Ning; Dai, Peng; Sun, Jianbin; Wang, Qin; Liang, Ping; Zhang, Wentao; Lei, Xiaoying; Yan, Zhen; Yang, Jing-Hua

2016-01-01

Viruses induce double-stranded RNA (dsRNA) in the host cells. The mammalian system has developed dsRNA-dependent recognition receptors such as RLRs that recognize the long stretches of dsRNA as PAMPs to activate interferon-mediated antiviral pathways and apoptosis in severe infection. Here we report an efficient antiviral immune response through dsRNA-dependent RLR receptor-mediated necroptosis against infections from different classes of viruses. We demonstrated that virus-infected A549 cells were efficiently killed in the presence of a chimeric RLR receptor, dsCARE. It measurably suppressed the interferon antiviral pathway but promoted IL-1β production. Canonical cell death analysis by morphologic assessment, phosphatidylserine exposure, caspase cleavage and chemical inhibition excluded the involvement of apoptosis and consistently suggested RLR receptor-mediated necroptosis as the underlying mechanism of infected cell death. The necroptotic pathway was augmented by the formation of RIP1-RIP3 necrosome, recruitment of MLKL protein and the activation of cathepsin D. Contributing roles of RIP1 and RIP3 were confirmed by gene knockdown. Furthermore, the necroptosis inhibitor necrostatin-1 but not the pan-caspase inhibitor zVAD impeded dsCARE-dependent infected cell death. Our data provides compelling evidence that the chimeric RLR receptor shifts the common interferon antiviral responses of infected cells to necroptosis and leads to rapid death of the virus-infected cells. This mechanism could be targeted as an efficient antiviral strategy. PMID:26935990

Integration of oxygen signaling at the consensus HRE.

PubMed

Wenger, Roland H; Stiehl, Daniel P; Camenisch, Gieri

2005-10-18

The hypoxia-inducible factor 1 (HIF-1) was initially identified as a transcription factor that regulated erythropoietin gene expression in response to a decrease in oxygen availability in kidney tissue. Subsequently, a family of oxygen-dependent protein hydroxylases was found to regulate the abundance and activity of three oxygen-sensitive HIFalpha subunits, which, as part of the HIF heterodimer, regulated the transcription of at least 70 different effector genes. In addition to responding to a decrease in tissue oxygenation, HIF is proactively induced, even under normoxic conditions, in response to stimuli that lead to cell growth, ultimately leading to higher oxygen consumption. The growing cell thus profits from an anticipatory increase in HIF-dependent target gene expression. Growth stimuli-activated signaling pathways that influence the abundance and activity of HIFs include pathways in which kinases are activated and pathways in which reactive oxygen species are liberated. These pathways signal to the HIF protein hydroxylases, as well as to HIF itself, by means of covalent or redox modifications and protein-protein interactions. The final point of integration of all of these pathways is the hypoxia-response element (HRE) of effector genes. Here, we provide comprehensive compilations of the known growth stimuli that promote increases in HIF abundance, of protein-protein interactions involving HIF, and of the known HIF effector genes. The consensus HRE derived from a comparison of the HREs of these HIF effectors will be useful for identification of novel HIF target genes, design of oxygen-regulated gene therapy, and prediction of effects of future drugs targeting the HIF system.

Role of Ficolin-A and Lectin Complement Pathway in the Innate Defense against Pathogenic Aspergillus Species

PubMed Central

Bidula, Stefan; Kenawy, Hany; Ali, Youssif M.; Sexton, Darren; Schwaeble, Wilhelm J.

2013-01-01

Aspergillus species are saprophytic molds causing life-threatening invasive fungal infections in the immunocompromised host. Innate immune recognition, in particular, the mechanisms of opsonization and complement activation, has been reported to be an integral part of the defense against fungi. We have shown that the complement component ficolin-A significantly binds to Aspergillus conidia and hyphae in a concentration-dependent manner and was inhibited by N-acetylglucosamine and N-acetylgalactosamine. Calcium-independent binding to Aspergillus fumigatus and A. terreus was observed, but binding to A. flavus and A. niger was calcium dependent. Ficolin-A binding to conidia was increased under low-pH conditions, and opsonization led to enhanced binding of conidia to A549 airway epithelial cells. In investigations of the lectin pathway of complement activation, ficolin-A-opsonized conidia did not lead to lectin pathway-specific C4 deposition. In contrast, the collectin mannose binding lectin C (MBL-C) but not MBL-A led to efficient lectin pathway activation on A. fumigatus in the absence of ficolin-A. In addition, ficolin-A opsonization led to a modulation of the proinflammatory cytokine interleukin-8. We conclude that ficolin-A may play an important role in the innate defense against Aspergillus by opsonizing conidia, immobilizing this fungus through enhanced adherence to epithelial cells and modulation of inflammation. However, it appears that other immune pattern recognition molecules, i.e., those of the collectin MBL-C, are involved in the Aspergillus-lectin complement pathway activation rather than ficolin-A. PMID:23478320

COT drives resistance to RAF inhibition through MAP kinase pathway reactivation.

PubMed

Johannessen, Cory M; Boehm, Jesse S; Kim, So Young; Thomas, Sapana R; Wardwell, Leslie; Johnson, Laura A; Emery, Caroline M; Stransky, Nicolas; Cogdill, Alexandria P; Barretina, Jordi; Caponigro, Giordano; Hieronymus, Haley; Murray, Ryan R; Salehi-Ashtiani, Kourosh; Hill, David E; Vidal, Marc; Zhao, Jean J; Yang, Xiaoping; Alkan, Ozan; Kim, Sungjoon; Harris, Jennifer L; Wilson, Christopher J; Myer, Vic E; Finan, Peter M; Root, David E; Roberts, Thomas M; Golub, Todd; Flaherty, Keith T; Dummer, Reinhard; Weber, Barbara L; Sellers, William R; Schlegel, Robert; Wargo, Jennifer A; Hahn, William C; Garraway, Levi A

2010-12-16

Oncogenic mutations in the serine/threonine kinase B-RAF (also known as BRAF) are found in 50-70% of malignant melanomas. Pre-clinical studies have demonstrated that the B-RAF(V600E) mutation predicts a dependency on the mitogen-activated protein kinase (MAPK) signalling cascade in melanoma-an observation that has been validated by the success of RAF and MEK inhibitors in clinical trials. However, clinical responses to targeted anticancer therapeutics are frequently confounded by de novo or acquired resistance. Identification of resistance mechanisms in a manner that elucidates alternative 'druggable' targets may inform effective long-term treatment strategies. Here we expressed ∼600 kinase and kinase-related open reading frames (ORFs) in parallel to interrogate resistance to a selective RAF kinase inhibitor. We identified MAP3K8 (the gene encoding COT/Tpl2) as a MAPK pathway agonist that drives resistance to RAF inhibition in B-RAF(V600E) cell lines. COT activates ERK primarily through MEK-dependent mechanisms that do not require RAF signalling. Moreover, COT expression is associated with de novo resistance in B-RAF(V600E) cultured cell lines and acquired resistance in melanoma cells and tissue obtained from relapsing patients following treatment with MEK or RAF inhibitors. We further identify combinatorial MAPK pathway inhibition or targeting of COT kinase activity as possible therapeutic strategies for reducing MAPK pathway activation in this setting. Together, these results provide new insights into resistance mechanisms involving the MAPK pathway and articulate an integrative approach through which high-throughput functional screens may inform the development of novel therapeutic strategies.

Ubiquitination and sumoylation of the HTLV-2 Tax-2B protein regulate its NF-κB activity: a comparative study with the HTLV-1 Tax-1 protein

PubMed Central

2012-01-01

Background Retroviruses HTLV-1 and HTLV-2 have homologous genomic structures but differ significantly in pathogenicity. HTLV-1 is associated with Adult T cell Leukemia (ATL), whereas infection by HTLV-2 has no association with neoplasia. Transformation of T lymphocytes by HTLV-1 is linked to the capacity of its oncoprotein Tax-1 to alter cell survival and cell cycle control mechanisms. Among these functions, Tax-1-mediated activation of cellular gene expression via the NF-κB pathway depends on Tax-1 post-translational modifications by ubiquitination and sumoylation. The Tax-2 protein of HTLV-2B (Tax-2B) is also modified by ubiquitination and sumoylation and activates the NF-κB pathway to a level similar to that of Tax-1. The present study aims to understand whether ubiquitination and sumoylation modifications are involved in Tax-2B-mediated activation of the NF-κB pathway. Results The comparison of Tax-1 and Tax-2B lysine to arginine substitution mutants revealed conserved patterns and levels of ubiquitination with notable difference in the lysine usage for sumoylation. Neither Tax-1 nor Tax-2B ubiquitination and sumoylation deficient mutants could activate the NF-κB pathway and fusion of ubiquitin or SUMO-1 to the C-terminus of the ubiquitination and sumoylation deficient Tax-2B mutant strikingly restored transcriptional activity. In addition, ubiquitinated forms of Tax-2B colocalized with RelA and IKKγ in prominent cytoplasmic structures associated with the Golgi apparatus, whereas colocalization of Tax-2B with the RelA subunit of NF-κB and the transcriptional coactivator p300 in punctate nuclear structures was dependent on Tax-2B sumoylation, as previously observed for Tax-1. Conclusions Both Tax-1 and Tax-2 activate the NF-κB pathway via similar mechanisms involving ubiquitination and sumoylation. Therefore, the different transforming potential of HTLV-1 and HTLV-2 is unlikely to be related to different modes of activation of the canonical NF-κB pathway. PMID:23217160

MAPK pathway activation by chronic lead-exposure increases vascular reactivity through oxidative stress/cyclooxygenase-2-dependent pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Simões, Maylla Ronacher, E-mail: yllars@hotmail.com; Department of Pharmacology, Universidad Autonoma de Madrid, Instituto de Investigación Hospital Universitario La Paz; Aguado, Andrea

Chronic exposure to low lead concentration produces hypertension; however, the underlying mechanisms remain unclear. We analyzed the role of oxidative stress, cyclooxygenase-2-dependent pathways and MAPK in the vascular alterations induced by chronic lead exposure. Aortas from lead-treated Wistar rats (1st dose: 10 μg/100 g; subsequent doses: 0.125 μg/100 g, intramuscular, 30 days) and cultured aortic vascular smooth muscle cells (VSMCs) from Sprague Dawley rats stimulated with lead (20 μg/dL) were used. Lead blood levels of treated rats attained 21.7 ± 2.38 μg/dL. Lead exposure increased systolic blood pressure and aortic ring contractile response to phenylephrine, reduced acetylcholine-induced relaxation and didmore » not affect sodium nitroprusside relaxation. Endothelium removal and L-NAME left-shifted the response to phenylephrine more in untreated than in lead-treated rats. Apocynin and indomethacin decreased more the response to phenylephrine in treated than in untreated rats. Aortic protein expression of gp91(phox), Cu/Zn-SOD, Mn-SOD and COX-2 increased after lead exposure. In cultured VSMCs lead 1) increased superoxide anion production, NADPH oxidase activity and gene and/or protein levels of NOX-1, NOX-4, Mn-SOD, EC-SOD and COX-2 and 2) activated ERK1/2 and p38 MAPK. Both antioxidants and COX-2 inhibitors normalized superoxide anion production, NADPH oxidase activity and mRNA levels of NOX-1, NOX-4 and COX-2. Blockade of the ERK1/2 and p38 signaling pathways abolished lead-induced NOX-1, NOX-4 and COX-2 expression. Results show that lead activation of the MAPK signaling pathways activates inflammatory proteins such as NADPH oxidase and COX-2, suggesting a reciprocal interplay and contribution to vascular dysfunction as an underlying mechanisms for lead-induced hypertension. - Highlights: • Lead-exposure increases oxidative stress, COX-2 expression and vascular reactivity. • Lead exposure activates MAPK signaling pathway. • ROS and COX-2 activation by MAPK in lead exposure • Relationship between vascular ROS and COX-2 products in lead exposure.« less

Autophagy regulates tissue overgrowth in a context-dependent manner.

PubMed

Pérez, E; Das, G; Bergmann, A; Baehrecke, E H

2015-06-01

Autophagy is a catabolic process that has been implicated both as a tumor suppressor and in tumor progression. Here, we investigate this dichotomy in cancer biology by studying the influence of altered autophagy in Drosophila models of tissue overgrowth. We find that the impact of altered autophagy depends on both genotype and cell type. As previously observed in mammals, decreased autophagy suppresses Ras-induced eye epithelial overgrowth. In contrast, autophagy restricts epithelial overgrowth in a Notch-dependent eye model. Even though decreased autophagy did not influence Hippo pathway-triggered overgrowth, activation of autophagy strongly suppresses this eye epithelial overgrowth. Surprisingly, activation of autophagy enhanced Hippo pathway-driven overgrowth in glia cells. These results indicate that autophagy has different influences on tissue growth in distinct contexts, and highlight the importance of understanding the influence of autophagy on growth to augment a rationale therapeutic strategy.

Carbohydrate Metabolism and Carbon Fixation in Roseobacter denitrificans OCh114

PubMed Central

Tang, Kuo-Hsiang; Feng, Xueyang; Tang, Yinjie J.; Blankenship, Robert E.

2009-01-01

The Roseobacter clade of aerobic marine proteobacteria, which compose 10–25% of the total marine bacterial community, has been reported to fix CO2, although it has not been determined what pathway is involved. In this study, we report the first metabolic studies on carbohydrate utilization, CO2 assimilation, and amino acid biosynthesis in the phototrophic Roseobacter clade bacterium Roseobacter denitrificans OCh114. We develop a new minimal medium containing defined carbon source(s), in which the requirements of yeast extract reported previously for the growth of R. denitrificans can be replaced by vitamin B12 (cyanocobalamin). Tracer experiments were carried out in R. denitrificans grown in a newly developed minimal medium containing isotopically labeled pyruvate, glucose or bicarbonate as a single carbon source or in combination. Through measurements of 13C-isotopomer labeling patterns in protein-derived amino acids, gene expression profiles, and enzymatic activity assays, we report that: (1) R. denitrificans uses the anaplerotic pathways mainly via the malic enzyme to fix 10–15% of protein carbon from CO2; (2) R. denitrificans employs the Entner-Doudoroff (ED) pathway for carbohydrate metabolism and the non-oxidative pentose phosphate pathway for the biosynthesis of histidine, ATP, and coenzymes; (3) the Embden-Meyerhof-Parnas (EMP, glycolysis) pathway is not active and the enzymatic activity of 6-phosphofructokinase (PFK) cannot be detected in R. denitrificans; and (4) isoleucine can be synthesized from both threonine-dependent (20% total flux) and citramalate-dependent (80% total flux) pathways using pyruvate as the sole carbon source. PMID:19794911

A signaling pathway contributing to platelet storage lesion development: targeting PI3-kinase–dependent Rap1 activation slows storage-induced platelet deterioration

PubMed Central

Schubert, Peter; Thon, Jonathan N.; Walsh, Geraldine M.; Chen, Cindy H.I.; Moore, Edwin D.; Devine, Dana V.; Kast, Juergen

2015-01-01

BACKGROUND The term platelet storage lesion (PSL) describes the structural and biochemical changes in platelets (PLTs) during storage. These are typified by alterations of morphologic features and PLT metabolism leading to reduced functionality and hence reduced viability for transfusion. While the manifestations of the storage lesion are well characterized, the biochemical pathways involved in the initiation of this process are unknown. STUDY DESIGN AND METHODS A complementary proteomic approach has recently been applied to analyze changes in the PLT proteome during storage. By employing stringent proteomic criteria, 12 proteins were identified as significantly and consistently changing in relative concentration over a 7-day storage period. Microscopy, Western blot analysis, flow cytometry, and PLT functionality analyses were used to unravel the involvement of a subset of these 12 proteins, which are connected through integrin signaling in one potential signaling pathway underlying storage lesion development. RESULTS Microscopic analysis revealed changes in localization of glycoprotein IIIa, Rap1, and talin during storage. Rap1 activation was observed to correlate with expression of the PLT activation marker CD62P. PLTs incubated for 7 days with the PI3-kinase inhibitor LY294002 showed diminished Rap1 activation as well as a moderate reduction in integrin αIIbβ3 activation and release of α-granules. Furthermore, this inhibitor seemed to improve PLT integrity and quality during storage as several in vitro probes showed a deceleration of PLT activation. CONCLUSION These results provide the first evidence for a signaling pathway mediating PSL in which PI3-kinase–dependent Rap1 activation leads to integrin αIIbβ3 activation and PLT degranulation. PMID:19497060

LPS Increases 5-LO Expression on Monocytes via an Activation of Akt-Sp1/NF-κB Pathways.

PubMed

Lee, Seung Jin; Seo, Kyo Won; Kim, Chi Dae

2015-05-01

5-Lipoxygenase (5-LO) plays a pivotal role in the progression of atherosclerosis. Therefore, this study investigated the molecular mechanisms involved in 5-LO expression on monocytes induced by LPS. Stimulation of THP-1 monocytes with LPS (0~3 µg/ml) increased 5-LO promoter activity and 5-LO protein expression in a concentration-dependent manner. LPS-induced 5-LO expression was blocked by pharmacological inhibition of the Akt pathway, but not by inhibitors of MAPK pathways including the ERK, JNK, and p38 MAPK pathways. In line with these results, LPS increased the phosphorylation of Akt, suggesting a role for the Akt pathway in LPS-induced 5-LO expression. In a promoter activity assay conducted to identify transcription factors, both Sp1 and NF-κB were found to play central roles in 5-LO expression in LPS-treated monocytes. The LPS-enhanced activities of Sp1 and NF-κB were attenuated by an Akt inhibitor. Moreover, the LPS-enhanced phosphorylation of Akt was significantly attenuated in cells pretreated with an anti-TLR4 antibody. Taken together, 5-LO expression in LPS-stimulated monocytes is regulated at the transcriptional level via TLR4/Akt-mediated activations of Sp1 and NF-κB pathways in monocytes.

African swine fever virus infects macrophages, the natural host cells, via clathrin- and cholesterol-dependent endocytosis.

PubMed

Galindo, Inmaculada; Cuesta-Geijo, Miguel Angel; Hlavova, Karolina; Muñoz-Moreno, Raquel; Barrado-Gil, Lucía; Dominguez, Javier; Alonso, Covadonga

2015-03-16

The main cellular target for African swine fever virus (ASFV) is the porcine macrophage. However, existing data about the early phases of infection were previously characterized in non-leukocyte cells such as Vero cells. Here, we report that ASFV enters the natural host cell using dynamin-dependent and clathrin-mediated endocytosis. This pathway is strongly pH-dependent during the first steps of infection in porcine macrophages. We investigated the effect of drugs inhibiting several endocytic pathways in macrophages and compared ASFV with vaccinia virus (VV), which apparently involves different entry pathways. The presence of cholesterol in cellular membranes was found to be essential for a productive ASFV infection while actin-dependent endocytosis and the participation of phosphoinositide-3-kinase (PI3K) activity were other cellular factors required in the process of viral entry. These findings improved our understanding of the ASFV interactions with macrophages that allow for successful viral replication. Copyright © 2015 Elsevier B.V. All rights reserved.

NIK and IKKbeta interdependence in NF-kappaB signalling--flux analysis of regulation through metabolites.

PubMed

Kim, Hong-Bum; Evans, Iona; Smallwood, Rod; Holcombe, Mike; Qwarnstrom, Eva E

2010-02-01

Activation of the transcription factor NF-kappaB is central to control of immune and inflammatory responses. Cytokine induced activation through the classical or canonical pathway relies on degradation of the inhibitor, IkappaBalpha and regulation by the IKKbeta kinase. In addition, the NF-kappaB is activated through the NF-kappaB-inducing kinase, NIK. Analysis of the IKK/NIK inter-relationship and its impact on NF-kappaB control, were analysed by mathematical modelling, using matrix formalism and stoichiometrically balanced reactions. The analysis considered a range of bio-reactions and core metabolites and their role in relation to kinase activation and in control of specific steps of the NF-kappaB pathway. The model predicts a growth-rate and time-dependent transfer of the primary kinase activity from IKKbeta to NIK. In addition, it suggests that NIK/IKKbeta interdependence is controlled by intermediates of phosphoribosylpyrophosphate (PRPP) within the glycolysis pathway, and thus, identifies a link between specific metabolic events and kinase activation in inflammatory signal transduction. Subsequent in vitro experiments, carried out to validate the impact of IKK/NIK interdependence, confirmed signal amplification at the level of the NF-kappaB/IkappaBalpha complex control in the presence of both kinases. Further, they demonstrate that the induced potentiation is due to synergistic enhancement of relA-dependent activation. Copyright (c) 2009 Elsevier Ireland Ltd. All rights reserved.

Oxygen activation by mononuclear nonheme iron dioxygenases involved in the degradation of aromatics.

PubMed

Wang, Yifan; Li, Jiasong; Liu, Aimin

2017-04-01

Molecular oxygen is utilized in numerous metabolic pathways fundamental for life. Mononuclear nonheme iron-dependent oxygenase enzymes are well known for their involvement in some of these pathways, activating O 2 so that oxygen atoms can be incorporated into their primary substrates. These reactions often initiate pathways that allow organisms to use stable organic molecules as sources of carbon and energy for growth. From the myriad of reactions in which these enzymes are involved, this perspective recounts the general mechanisms of aromatic dihydroxylation and oxidative ring cleavage, both of which are ubiquitous chemical reactions found in life-sustaining processes. The organic substrate provides all four electrons required for oxygen activation and insertion in the reactions mediated by extradiol and intradiol ring-cleaving catechol dioxygenases. In contrast, two of the electrons are provided by NADH in the cis-dihydroxylation mechanism of Rieske dioxygenases. The catalytic nonheme Fe center, with the aid of active site residues, facilitates these electron transfers to O 2 as key elements of the activation processes. This review discusses some general questions for the catalytic strategies of oxygen activation and insertion into aromatic compounds employed by mononuclear nonheme iron-dependent dioxygenases. These include: (1) how oxygen is activated, (2) whether there are common intermediates before oxygen transfer to the aromatic substrate, and (3) are these key intermediates unique to mononuclear nonheme iron dioxygenases?

Low-dose gamma-ray irradiation induces translocation of Nrf2 into nuclear in mouse macrophage RAW264.7 cells.

PubMed

Tsukimoto, Mitsutoshi; Tamaishi, Nana; Homma, Takujiro; Kojima, Shuji

2010-01-01

The transcription factor nuclear erythroid-derived 2-related factor 2 (Nrf2) regulates expression of genes encoding antioxidant proteins involved in cellular redox homeostasis, while gamma-ray irradiation is known to induce reactive oxygen species in vivo. Although activation of Nrf2 by various stresses has been studied, it has not yet been determined whether ionizing irradiation induces activation of Nrf2. Therefore, we investigated activation of Nrf2 in response to gamma-irradiation in mouse macrophage RAW264.7 cells. Irradiation of cells with gamma-rays induced an increase of Nrf2 expression. Even 0.1 Gy of gamma-irradiation induced a translocation of Nrf2 from cytoplasm to the nucleus, indicating the activation of Nrf2 by low-dose irradiation. Expression of heme oxygenase-1, which is regulated by Nrf2, was also increased at 24 h after irradiation with more than 0.1 Gy of gamma-rays. Furthermore, the activation of Nrf2 was suppressed by U0126, which is an inhibitor of the extracellular signal regulated protein kinase 1/2 (ERK1/2) pathway, suggesting involvement of ERK1/2-dependent pathway in the irradiation-induced activation of Nrf2. Our results indicate that low-dose gamma-irradiation induces activation of Nrf2 through ERK1/2-dependent pathways.

Echicetin Coated Polystyrene Beads: A Novel Tool to Investigate GPIb-Specific Platelet Activation and Aggregation

PubMed Central

Petunin, Alexey; Clemetson, Kenneth J.; Gambaryan, Stepan; Walter, Ulrich

2014-01-01

von Willebrand factor/ristocetin (vWF/R) induces GPIb-dependent platelet agglutination and activation of αIIbβ3 integrin, which also binds vWF. These conditions make it difficult to investigate GPIb-specific signaling pathways in washed platelets. Here, we investigated the specific mechanisms of GPIb signaling using echicetin-coated polystyrene beads, which specifically activate GPIb. We compared platelet activation induced by echicetin beads to vWF/R. Human platelets were stimulated with polystyrene beads coated with increasing amounts of echicetin and platelet activation by echicetin beads was then investigated to reveal GPIb specific signaling. Echicetin beads induced αIIbβ3-dependent aggregation of washed platelets, while under the same conditions vWF/R treatment led only to αIIbβ3-independent platelet agglutination. The average distance between the echicetin molecules on the polystyrene beads must be less than 7 nm for full platelet activation, while the total amount of echicetin used for activation is not critical. Echicetin beads induced strong phosphorylation of several proteins including p38, ERK and PKB. Synergistic signaling via P2Y12 and thromboxane receptor through secreted ADP and TxA2, respectively, were important for echicetin bead triggered platelet activation. Activation of PKG by the NO/sGC/cGMP pathway inhibited echicetin bead-induced platelet aggregation. Echicetin-coated beads are powerful and reliable tools to study signaling in human platelets activated solely via GPIb and GPIb-triggered pathways. PMID:24705415

Activity-Dependent Inhibitory Gating in Molecular Signaling Cascades Induces a Novel Form of Intermediate-Term Synaptic Facilitation in "Aplysia Californica"

ERIC Educational Resources Information Center

Fischbach, Soren; Kopec, Ashley M.; Carew, Thomas J.

2014-01-01

Mechanistically distinct forms of long-lasting plasticity and memory can be induced by a variety of different training patterns. Although several studies have identified distinct molecular pathways that are engaged during these different training patterns, relatively little work has explored potential interactions between pathways when they are…

Study on the apoptosis mediated by cytochrome c and factors that affect the activation of bovine longissimus muscle during postmortem aging.

PubMed

Zhang, Jiaying; Yu, Qunli; Han, Ling; Chen, Cheng; Li, Hang; Han, Guangxing

2017-06-01

This study investigates whether bovine longissimus muscle cell apoptosis occurs during postmortem aging and whether apoptosis is dependent on the mitochondria pathway. This study also determines the apoptosis process mediated by cytochrome c after its release from mitochondria and the factors that affect the activation processes. Results indicate that apoptotic nuclei were detected at 12 h postmortem. Cytochrome c release from the mitochondria to the cytoplasm activated the caspase-9 and caspase-3 at early postmortem aging and the activation of caspase-9 occurs before the activation of caspase-3. The pH level decreased during the first 48 h postmortem, whereas the mitochondria membrane permeability increased from 6 to 12 h. Results demonstrate that an apoptosis process of bovine muscle occurred during postmortem aging. Apoptosis was dependent on the mitochondria pathway and occurred at early postmortem aging. Increased mitochondria membrane permeability and low pH are necessary conditions for the release of cytochrome c during postmortem aging.

Calcium and Superoxide-Mediated Pathways Converge to Induce Nitric Oxide-Dependent Apoptosis in Mycobacterium fortuitum-Infected Fish Macrophages.

PubMed

Datta, Debika; Khatri, Preeti; Banerjee, Chaitali; Singh, Ambika; Meena, Ramavatar; Saha, Dhira Rani; Raman, Rajagopal; Rajamani, Paulraj; Mitra, Abhijit; Mazumder, Shibnath

2016-01-01

Mycobacterium fortuitum causes 'mycobacteriosis' in wide range of hosts although the mechanisms remain largely unknown. Here we demonstrate the role of calcium (Ca+2)-signalling cascade on M. fortuitum-induced apoptosis in headkidney macrophages (HKM) of Clarias sp. M. fortuitum could trigger intracellular-Ca+2 influx leading to the activation of calmodulin (CaM), protein kinase C alpha (PKCα) and Calmodulin kinase II gamma (CaMKIIg). Gene silencing and inhibitor studies established the role of CaM in M. fortuitum pathogenesis. We noted that CaMKIIg activation is regulated by CaM as well as PKCα-dependent superoxide anions. This is altogether first report of oxidised CaMKIIg in mycobacterial infections. Our studies with targeted-siRNA and pharmacological inhibitors implicate CaMKIIg to be pro-apoptotic and critical for the activation of extra-cellular signal regulated kinase 1/2 (ERK1/2). Inhibiting the ERK1/2 pathway attenuated nitric oxide synthase 2 (NOS2)-induced nitric oxide (NO) production. Conversely, inhibiting the NOS2-NO axis by specific-siRNA and inhibitors down-regulated ERK1/2 activation suggesting the crosstalk between ERK1/2 and NO is essential for pathogenesis induced by the bacterium. Silencing the NOS2-NO axis enhanced intracellular bacterial survival and attenuated caspase-8 mediated activation of caspase-3 in the infected HKM. Our findings unveil hitherto unknown mechanism of M. fortuitum pathogenesis. We propose that M. fortuitum triggers intracellular Ca+2 elevations resulting in CaM activation and PKCα-mediated superoxide generation. The cascade converges in common pathway mediated by CaMKIIg resulting in the activation of ERK1/2-NOS2 axis. The crosstalk between ERK1/2 and NO shifts the balance in favour of caspase dependent apoptosis of M. fortuitum-infected HKM.

Caveolin 1-related autophagy initiated by aldosterone-induced oxidation promotes liver sinusoidal endothelial cells defenestration.

PubMed

Luo, Xiaoying; Dan Wang; Luo, Xuan; Zhu, Xintao; Wang, Guozhen; Ning, Zuowei; Li, Yang; Ma, Xiaoxin; Yang, Renqiang; Jin, Siyi; Huang, Yun; Meng, Ying; Li, Xu

2017-10-01

Aldosterone, with pro-oxidation and pro-autophagy capabilities, plays a key role in liver fibrosis. However, the mechanisms underlying aldosterone-promoted liver sinusoidal endothelial cells (LSECs) defenestration remain unknown. Caveolin 1 (Cav1) displays close links with autophagy and fenestration. Hence, we aim to investigate the role of Cav1-related autophagy in LSECs defenestration. We found the increase of aldosterone/MR (mineralocorticoid receptor) level, oxidation, autophagy, and defenestration in LSECs in the human fibrotic liver, BDL or hyperaldosteronism models; while antagonizing aldosterone or inhibiting autophagy relieved LSECs defenestration in BDL-induced fibrosis or hyperaldosteronism models. In vitro, fenestrae of primary LSECs gradually shrank, along with the down-regulation of the NO-dependent pathway and the augment of the AMPK-dependent autophagy; these effects were aggravated by rapamycin (an autophagy activator) or aldosterone treatment. Additionally, aldosterone increased oxidation mediated by Cav1, reduced ATP generation, and subsequently induced the AMPK-dependent autophagy, leading to the down-regulation of the NO-dependent pathway and LSECs defenestration. These effects were reversed by MR antagonist spironolactone, antioxidants or autophagy inhibitors. Besides, aldosterone enhanced the co-immunoprecipitation of Cav1 with p62 and ubiquitin, and induced Cav1 co-immunofluorescence staining with LC3, ubiquitin, and F-actin in the perinuclear area of LSECs. Furthermore, aldosterone treatment increased the membrane protein level of Cav1, whereas decrease the cytoplasmic protein level of Cav1, indicating that aldosterone induced Cav1-related selective autophagy and F-actin remodeling to promote defenestration. Consequently, Cav1-related selective autophagy initiated by aldosterone-induced oxidation promotes LSECs defenestration via activating the AMPK-ULK1 pathway and inhibiting the NO-dependent pathway. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Multivariate inference of pathway activity in host immunity and response to therapeutics

PubMed Central

Goel, Gautam; Conway, Kara L.; Jaeger, Martin; Netea, Mihai G.; Xavier, Ramnik J.

2014-01-01

Developing a quantitative view of how biological pathways are regulated in response to environmental factors is central for understanding of disease phenotypes. We present a computational framework, named Multivariate Inference of Pathway Activity (MIPA), which quantifies degree of activity induced in a biological pathway by computing five distinct measures from transcriptomic profiles of its member genes. Statistical significance of inferred activity is examined using multiple independent self-contained tests followed by a competitive analysis. The method incorporates a new algorithm to identify a subset of genes that may regulate the extent of activity induced in a pathway. We present an in-depth evaluation of specificity, robustness, and reproducibility of our method. We benchmarked MIPA's false positive rate at less than 1%. Using transcriptomic profiles representing distinct physiological and disease states, we illustrate applicability of our method in (i) identifying gene–gene interactions in autophagy-dependent response to Salmonella infection, (ii) uncovering gene–environment interactions in host response to bacterial and viral pathogens and (iii) identifying driver genes and processes that contribute to wound healing and response to anti-TNFα therapy. We provide relevant experimental validation that corroborates the accuracy and advantage of our method. PMID:25147207

A switch in the mode of Wnt signaling orchestrates the formation of germline stem cell differentiation niche in Drosophila

PubMed Central

Upadhyay, Maitreyi; Kuna, Michael; Tudor, Sara; Martino Cortez, Yesenia

2018-01-01

Germline stem cell (GSC) self-renewal and differentiation into gametes is regulated by both intrinsic factors in the germ line as well as extrinsic factors from the surrounding somatic niche. dWnt4, in the escort cells of the adult somatic niche promotes GSC differentiation using the canonical β-catenin-dependent transcriptional pathway to regulate escort cell survival, adhesion to the germ line and downregulation of self-renewal signaling. Here, we show that in addition to the β-catenin-dependent canonical pathway, dWnt4 also uses downstream components of the Wnt non-canonical pathway to promote escort cell function earlier in development. We find that the downstream non-canonical components, RhoA, Rac1 and cdc42, are expressed at high levels and are active in escort cell precursors of the female larval gonad compared to the adult somatic niche. Consistent with this expression pattern, we find that the non-canonical pathway components function in the larval stages but not in adults to regulate GSC differentiation. In the larval gonad, dWnt4, RhoA, Rac1 and cdc42 are required to promote intermingling of escort cell precursors, a function that then promotes proper escort cell function in the adults. We find that dWnt4 acts by modulating the activity of RhoA, Rac1 and cdc42, but not their protein levels. Together, our results indicate that at different points of development, dWnt4 switches from using the non-canonical pathway components to using a β-catenin-dependent canonical pathway in the escort cells to facilitate the proper differentiation of GSCs. PMID:29370168

Hexavalent Chromium Cr(VI) Up-Regulates COX-2 Expression through an NFκB/c-Jun/AP-1–Dependent Pathway

PubMed Central

Zuo, Zhenghong; Cai, Tongjian; Li, Jingxia; Zhang, Dongyun; Yu, Yonghui

2012-01-01

Background: Hexavalent chromium [Cr(VI)] is recognized as a human carcinogen via inhalation. However, the molecular mechanisms by which Cr(VI) causes cancers are not well understood. Objectives: We evaluated cyclooxygenase-2 (COX-2) expression and the signaling pathway leading to this induction due to Cr(VI) exposure in cultured cells. Methods: We used the luciferase reporter assay and Western blotting to determine COX-2 induction by Cr(VI). We used dominant negative mutant, genetic knockout, gene knockdown, and chromatin immunoprecipitation approaches to elucidate the signaling pathway leading to COX-2 induction. Results: We found that Cr(VI) exposure induced COX-2 expression in both normal human bronchial epithelial cells and mouse embryonic fibroblasts in a concentration- and time-dependent manner. Deletion of IKKβ [inhibitor of transcription factor NFκB (IκB) kinase β; an upstream kinase responsible for nuclear factor κB (NFκB) activation] or overexpression of TAM67 (a dominant-negative mutant of c-Jun) dramatically inhibited the COX-2 induction due to Cr(VI), suggesting that both NFκB and c-Jun/AP-1 pathways were required for Cr(VI)-induced COX-2 expression. Our results show that p65 and c-Jun are two major components involved in NFκB and AP-1 activation, respectively. Moreover, our studies suggest crosstalk between NFκB and c-Jun/AP-1 pathways in cellular response to Cr(VI) exposure for COX-2 induction. Conclusion: We demonstrate for the first time that Cr(VI) is able to induce COX-2 expression via an NFκB/c-Jun/AP-1–dependent pathway. Our results provide novel insight into the molecular mechanisms linking Cr(VI) exposure to lung inflammation and carcinogenesis. PMID:22472290

RIM101-Dependent and -Independent Pathways Govern pH Responses in Candida albicans

PubMed Central

Davis, Dana; Wilson, R. Bryce; Mitchell, Aaron P.

2000-01-01

Growth and differentiation of Candida albicans over a broad pH range underlie its ability to infect an array of tissues in susceptible hosts. We identified C. albicans RIM101, RIM20, and RIM8 based on their homology to components of the one known fungal pH response pathway. PCR product-disruption mutations in each gene cause defects in three responses to alkaline pH: filamentation, induction of PRA1 and PHR1, and repression of PHR2. We find that RIM101 itself is an alkaline-induced gene that also depends on Rim20p and Rim8p for induction. Two observations indicate that a novel pH response pathway also exists. First, PHR2 becomes an alkaline-induced gene in the absence of Rim101p, Rim20p, or Rim8p. Second, we created strains in which Rim101p activity is independent of Rim20p and Rim8p; in these strains, filamentation remains pH dependent. Thus, pH governs gene expression and cellular differentiation in C. albicans through both RIM101-dependent and RIM101-independent pathways. PMID:10629054

Arabidopsis MYC Transcription Factors Are the Target of Hormonal Salicylic Acid/Jasmonic Acid Cross Talk in Response to Pieris brassicae Egg Extract1[OPEN

PubMed Central

Schmiesing, André; Gouhier-Darimont, Caroline

2016-01-01

Arabidopsis (Arabidopsis thaliana) plants recognize insect eggs and activate the salicylic acid (SA) pathway. As a consequence, expression of defense genes regulated by the jasmonic acid (JA) pathway is suppressed and larval performance is enhanced. Cross talk between defense signaling pathways is common in plant-pathogen interactions, but the molecular mechanism mediating this phenomenon is poorly understood. Here, we demonstrate that egg-induced SA/JA antagonism works independently of the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor ORA59, which controls the ERF branch of the JA pathway. In addition, treatment with egg extract did not enhance expression or stability of JASMONATE ZIM-domain transcriptional repressors, and SA/JA cross talk did not involve JASMONATE ASSOCIATED MYC2-LIKEs, which are negative regulators of the JA pathway. Investigating the stability of MYC2, MYC3, and MYC4, three basic helix-loop-helix transcription factors that additively control jasmonate-related defense responses, we found that egg extract treatment strongly diminished MYC protein levels in an SA-dependent manner. Furthermore, we identified WRKY75 as a novel and essential factor controlling SA/JA cross talk. These data indicate that insect eggs target the MYC branch of the JA pathway and uncover an unexpected modulation of SA/JA antagonism depending on the biological context in which the SA pathway is activated. PMID:26884488

Arabidopsis MYC Transcription Factors Are the Target of Hormonal Salicylic Acid/Jasmonic Acid Cross Talk in Response to Pieris brassicae Egg Extract.

PubMed

Schmiesing, André; Emonet, Aurélia; Gouhier-Darimont, Caroline; Reymond, Philippe

2016-04-01

Arabidopsis (Arabidopsis thaliana) plants recognize insect eggs and activate the salicylic acid (SA) pathway. As a consequence, expression of defense genes regulated by the jasmonic acid (JA) pathway is suppressed and larval performance is enhanced. Cross talk between defense signaling pathways is common in plant-pathogen interactions, but the molecular mechanism mediating this phenomenon is poorly understood. Here, we demonstrate that egg-induced SA/JA antagonism works independently of the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor ORA59, which controls the ERF branch of the JA pathway. In addition, treatment with egg extract did not enhance expression or stability of JASMONATE ZIM-domain transcriptional repressors, and SA/JA cross talk did not involve JASMONATE ASSOCIATED MYC2-LIKEs, which are negative regulators of the JA pathway. Investigating the stability of MYC2, MYC3, and MYC4, three basic helix-loop-helix transcription factors that additively control jasmonate-related defense responses, we found that egg extract treatment strongly diminished MYC protein levels in an SA-dependent manner. Furthermore, we identified WRKY75 as a novel and essential factor controlling SA/JA cross talk. These data indicate that insect eggs target the MYC branch of the JA pathway and uncover an unexpected modulation of SA/JA antagonism depending on the biological context in which the SA pathway is activated. © 2016 American Society of Plant Biologists. All Rights Reserved.

Design and functional characterization of a novel, arrestin-biased designer G protein-coupled receptor.

PubMed

Nakajima, Ken-ichiro; Wess, Jürgen

2012-10-01

Mutational modification of distinct muscarinic receptor subtypes has yielded novel designer G protein-coupled receptors (GPCRs) that are unable to bind acetylcholine (ACh), the endogenous muscarinic receptor ligand, but can be efficiently activated by clozapine-N-oxide (CNO), an otherwise pharmacologically inert compound. These CNO-sensitive designer GPCRs [alternative name: designer receptors exclusively activated by designer drug (DREADDs)] have emerged as powerful new tools to dissect the in vivo roles of distinct G protein signaling pathways in specific cell types or tissues. As is the case with other GPCRs, CNO-activated DREADDs not only couple to heterotrimeric G proteins but can also recruit proteins of the arrestin family (arrestin-2 and -3). Accumulating evidence suggests that arrestins can act as scaffolding proteins to promote signaling through G protein-independent signaling pathways. To explore the physiological relevance of these arrestin-dependent signaling pathways, the availability of an arrestin-biased DREADD would be highly desirable. In this study, we describe the development of an M₃ muscarinic receptor-based DREADD [Rq(R165L)] that is no longer able to couple to G proteins but can recruit arrestins and promote extracellular signal-regulated kinase-1/2 phosphorylation in an arrestin- and CNO-dependent fashion. Moreover, CNO treatment of mouse insulinoma (MIN6) cells expressing the Rq(R165L) construct resulted in a robust, arrestin-dependent stimulation of insulin release, directly implicating arrestin signaling in the regulation of insulin secretion. This newly developed arrestin-biased DREADD represents an excellent novel tool to explore the physiological relevance of arrestin signaling pathways in distinct tissues and cell types.

Role of CSL-dependent and independent Notch signaling pathways in cell apoptosis.

PubMed

Zeng, Chong; Xing, Rui; Liu, Jing; Xing, Feiyue

2016-01-01

Apoptosis is a normally biological phenomenon in various organisms, involving complexly molecular mechanisms with a series of signaling processes. Notch signaling is found evolutionarily conserved in many species, playing a critical role in embryonic development, normal tissue homeostasis, angiogenesis and immunoregulation. The focus of this review is on currently novel advances about roles of CSL-dependent and independent Notch signaling pathways in cell apoptosis. The CSL can bind Notch intracellular domain (NIC) to act as a switch in mediating transcriptional activation or inactivation of the Notch signaling pathway downstream genes in the nucleus. It shows that CSL-dependent signaling regulates the cell apoptosis through Hes-1-PTEN-AKT-mTOR signaling, but rather the CSL-independent signaling mediates the cell apoptosis possibly via NIC-mTORC2-AKT-mTOR signaling, providing a new insight into apoptotic mechanisms.

Betalactam antibiotics affect human dendritic cells maturation through MAPK/NF-kB systems. Role in allergic reactions to drugs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lopez, Soledad; Department of Medical Biochemistry, Molecular Biology and Immunology, The University of Seville Medical School, Seville; Gomez, Enrique

The mechanisms leading to drug allergy in predisposed patients, especially those related to T-cell-mediated drug hypersensitivity, are not well understood. A key event in allergic reactions to drugs is the maturation process undergone by dendritic cells (DCs). Although amoxicillin (AX) has been reported to interact and maturate DCs from patients with AX-induced delayed-type hypersensitivity, the cell signaling pathways related to AX-mediated DC maturation have not been elucidated. We sought to determine the role of the MAPK and NF-κΒ pathways on AX-induced DC maturation and functional status. For that purpose, in monocyte-derived-DCs from AX-delayed allergic patients and tolerant subjects, we analyzedmore » the activation pattern of p38MAPK, JNK, and ERK signaling and the NF-κB, maturation markers as well as endocytosis and allostimulatory capacities driven by AX-stimulated-DCs. Our data reveal that AX induces an increase in the phosphorylation levels of the three MAPKsand activated NF-κB in DCs from allergic patients. Moreover, the inhibition of these pathways prevents the up-regulation of surface molecules induced by AX. Additionally, we observed that the allostimulatory capacity and the endocytosis down-regulation in AX-stimulated-DCs from allergic patients depend on JNK and NF-κB activities. Taken together, our data shed light for the first time on the main signaling pathways involved in DC maturation from AX-delayed allergic patient. - Highlights: • The cell signaling pathways related to drug-mediated DC maturation were tested. • Amoxicillin induces activation of MAPK and NF-κB in DCs from allergic patients. • The inhibition of these pathways prevents the up-regulation of DC surface molecules. • Their allostimulatory and endocytosis capacities depend on JNK and NF-κB activities. • The low involvement of p38-MAPK could be the cause of an incomplete DC maturation.« less

Enhanced migration of tissue inhibitor of metalloproteinase overexpressing hepatoma cells is attributed to gelatinases: Relevance to intracellular signaling pathways

PubMed Central

Roeb, Elke; Bosserhoff, Anja-Katrin; Hamacher, Sabine; Jansen, Bettina; Dahmen, Judith; Wagner, Sandra; Matern, Siegfried

2005-01-01

AIM: To study the effect of gelatinases (especially MMP-9) on migration of tissue inhibitor of metalloproteinase (TIMP-1) overexpressing hepatoma cells. METHODS: Wild type HepG2 cells, cells stably transfected with TIMP-1 and TIMP-1 antagonist (MMP-9-H401A, a catalytically inactive matrix metalloproteinase (MMP) which still binds and neutralizes TIMP-1) were incubated in Boyden chambers either with or without Galardin (a synthetic inhibitor of MMP-1, -2, -3, -8, -9) or a specific inhibitor of gelatinases. RESULTS: Compared to wild type HepG2 cells, the cells overexpressing TIMP-1 showed 115% migration (P<0.05) and the cells overexpressing MMP-9-H401A showed 62% migration (P<0.01). Galardin reduced cell migration dose dependently in all cases. The gelatinase inhibitor reduced migration in TIMP-1 overexpressing cells predominantly. Furthermore, we examined intracellular signal transduction pathways of TIMP-1-dependent HepG2 cells. TIMP-1 deactivates cell signaling pathways of MMP-2 and MMP-9 involving p38 mitogen-activated protein kinase. Specific blockade of the ERK pathway suppresses gelatinase expression either in the presence or absence of TIMP-1. CONCLUSION: Overexpressing functional TIMP-1- enhanced migration of HepG2-TIMP-1 cells depends on enhanced MMP-activity, especially MMP-9. PMID:15754388

Atypical regulators of Wnt/β-catenin signaling as potential therapeutic targets in Hepatocellular Carcinoma.

PubMed

Chen, Jianxiang; Rajasekaran, Muthukumar; Hui, Kam M

2017-06-01

Hepatocellular carcinoma is one of the most common causes of cancer-related death worldwide. Hepatocellular carcinoma development depends on the inhibition and activation of multiple vital pathways, including the Wnt signaling pathway. The Wnt/β-catenin pathway lies at the center of various signaling pathways that regulate embryonic development, tissue homeostasis and cancers. Activation of the Wnt/β-catenin pathway has been observed frequently in hepatocellular carcinoma. However, activating mutations in β-catenin, Axin and Adenomatous Polyposis Coli only contribute to a portion of the Wnt signaling hyper-activation observed in hepatocellular carcinoma. Therefore, besides mutations in the canonical Wnt components, there must be additional atypical regulation or regulators during Wnt signaling activation that promote liver carcinogenesis. In this mini-review, we have tried to summarize some of these well-established factors and to highlight some recently identified novel factors in the Wnt/β-catenin signaling pathway in hepatocellular carcinoma. Impact statement Early recurrence of human hepatocellular carcinoma (HCC) is a frequent cause of poor survival after potentially curative liver resection. Among the deregulated signaling cascades in HCC, evidence indicates that alterations in the Wnt/β-catenin signaling pathway play key roles in hepatocarcinogenesis. In this review, we summarize the potential molecular mechanisms how the microtubule-associated Protein regulator of cytokinesis 1 (PRC1), a direct Wnt signaling target previously identified in our laboratory to be up-regulated in HCC, in promoting cancer proliferation, stemness, metastasis and tumorigenesis through a complex regulatory circuitry of Wnt3a activities.

HIV-1 Rev protein specifies the viral RNA export pathway by suppressing TAP/NXF1 recruitment

PubMed Central

Taniguchi, Ichiro; Mabuchi, Naoto; Ohno, Mutsuhito

2014-01-01

Nuclear RNA export pathways in eukaryotes are often linked to the fate of a given RNA. Therefore, the choice of export pathway should be well-controlled to avoid an unfavorable effect on gene expression. Although some RNAs could be exported by more than one pathway, little is known about how the choice is regulated. This issue is highlighted when the human immunodeficiency virus type 1 (HIV-1) Rev protein induces the export of singly spliced and unspliced HIV-1 transcripts. How these RNAs are exported is not well understood because such transcripts should have the possibility of utilizing CRM1-dependent export via Rev or cellular TAP/NXF1-dependent export via the transcription/export (TREX) complex, or both. Here we found that Rev suppressed TAP/NXF1-dependent export of model RNA substrates that recapitulated viral transcripts. In this effect, Rev interacted with the cap-binding complex and inhibited the recruitment of the TREX complex. Thus, Rev controls the identity of the factor occupying the cap-proximal region that determines the RNA export pathway. This ribonucleoprotein remodeling activity of Rev may favor viral gene expression. PMID:24753416

Clathrin-independent internalization and recycling

PubMed Central

Gong, Qiang; Huntsman, Christopher; Ma, Dzwokai

2008-01-01

Abstract The functionality of receptor and channel proteins depends directly upon their expression level on the plasma membrane. Therefore, the ability to selectively adjust the surface level of a particular receptor or channel protein is pivotal to many cellular signalling events. The internalization and recycling pathway plays a major role in the regulation of protein surface level, and thus has been a focus of research for many years. Although several endocytic pathways have been identified, most of our knowledge has come from the clathrin-dependent pathway, while the other pathways remain much less well defined. Considering that clathrin-independent internalization may account for as much as 50% of the total endocytic activity in the cell, the lack of such knowledge constitutes a major gap in our efforts to understand how different internalization pathways are utilized and co-ordinated. Recent studies have provided valuable insights into this area, yet many more questions still remain. In this review, we will give a panoramic introduction to the current knowledge of various internalization and recycling pathways, with an emphasis on the latest findings that have broadened our view of the clathrin-independent pathways. We will also dedicate one section to the emerging studies of the clathrin-independent internalization pathways in neuronal cells. PMID:18039352

A systematic genetic screen for genes involved in sensing inorganic phosphate availability in Saccharomyces cerevisiae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Joonhyuk; Rajagopal, Abbhirami; Xu, Yi -Fan

Saccharomyces cerevisiae responds to changes in extracellular inorganic phosphate (Pi) availability by regulating the activity of the phosphate-responsive (PHO) signaling pathway, enabling cells to maintain intracellular levels of the essential nutrient P i. P i-limitation induces upregulation of inositol heptakisphosphate (IP 7) synthesized by the inositol hexakisphosphate kinase Vip1, triggering inhibition of the Pho80/Pho85 cyclin-cyclin dependent kinase (CDK) complex by the CDK inhibitor Pho81, which upregulates the PHO regulon through the CDK target and transcription factor Pho4. To identify genes that are involved in signaling upstream of the Pho80/Pho85/Pho81 complex and how they interact with each other to regulate themore » PHO pathway, we performed genome-wide screens with the synthetic genetic array method. We identified more than 300 mutants with defects in signaling upstream of the Pho80/Pho85/Pho81 complex, including AAH1, which encodes an adenine deaminase that negatively regulates the PHO pathway in a Vip1-dependent manner. Moreover, we showed that even in the absence of VIP1, the PHO pathway can be activated under prolonged periods of P i starvation, suggesting complexity in the mechanisms by which the PHO pathway is regulated.« less

A systematic genetic screen for genes involved in sensing inorganic phosphate availability in Saccharomyces cerevisiae

DOE PAGES

Choi, Joonhyuk; Rajagopal, Abbhirami; Xu, Yi -Fan; ...

2017-05-17

Saccharomyces cerevisiae responds to changes in extracellular inorganic phosphate (Pi) availability by regulating the activity of the phosphate-responsive (PHO) signaling pathway, enabling cells to maintain intracellular levels of the essential nutrient P i. P i-limitation induces upregulation of inositol heptakisphosphate (IP 7) synthesized by the inositol hexakisphosphate kinase Vip1, triggering inhibition of the Pho80/Pho85 cyclin-cyclin dependent kinase (CDK) complex by the CDK inhibitor Pho81, which upregulates the PHO regulon through the CDK target and transcription factor Pho4. To identify genes that are involved in signaling upstream of the Pho80/Pho85/Pho81 complex and how they interact with each other to regulate themore » PHO pathway, we performed genome-wide screens with the synthetic genetic array method. We identified more than 300 mutants with defects in signaling upstream of the Pho80/Pho85/Pho81 complex, including AAH1, which encodes an adenine deaminase that negatively regulates the PHO pathway in a Vip1-dependent manner. Moreover, we showed that even in the absence of VIP1, the PHO pathway can be activated under prolonged periods of P i starvation, suggesting complexity in the mechanisms by which the PHO pathway is regulated.« less

Reciprocal feedback regulation of PI3K and androgen receptor signaling in PTEN-deficient prostate cancer.

PubMed

Carver, Brett S; Chapinski, Caren; Wongvipat, John; Hieronymus, Haley; Chen, Yu; Chandarlapaty, Sarat; Arora, Vivek K; Le, Carl; Koutcher, Jason; Scher, Howard; Scardino, Peter T; Rosen, Neal; Sawyers, Charles L

2011-05-17

Prostate cancer is characterized by its dependence on androgen receptor (AR) and frequent activation of PI3K signaling. We find that AR transcriptional output is decreased in human and murine tumors with PTEN deletion and that PI3K pathway inhibition activates AR signaling by relieving feedback inhibition of HER kinases. Similarly, AR inhibition activates AKT signaling by reducing levels of the AKT phosphatase PHLPP. Thus, these two oncogenic pathways cross-regulate each other by reciprocal feedback. Inhibition of one activates the other, thereby maintaining tumor cell survival. However, combined pharmacologic inhibition of PI3K and AR signaling caused near-complete prostate cancer regressions in a Pten-deficient murine prostate cancer model and in human prostate cancer xenografts, indicating that both pathways coordinately support survival. Copyright © 2011 Elsevier Inc. All rights reserved.

Interplay between adenylate metabolizing enzymes and amp-activated protein kinase.

PubMed

Camici, Marcella; Allegrini, Simone; Tozzi, Maria Grazia

2018-05-18

Purine nucleotides are involved in a variety of cellular functions, such as energy storage and transfer, and signalling, in addition to being the precursors of nucleic acids and cofactors of many biochemical reactions. They can be generated through two separate pathways, the de novo biosynthesis pathway and the salvage pathway. De novo purine biosynthesis leads to the formation of IMP, from which the adenylate and guanylate pools are generated by two additional steps. The salvage pathways utilize hypoxanthine, guanine and adenine to generate the corresponding mononucleotides. Despite several decades of research on the subject, new and surprising findings on purine metabolism are constantly being reported, and some aspects still need to be elucidated. Recently, purine biosynthesis has been linked to the metabolic pathways regulated by AMP-activated protein kinase (AMPK). AMPK is the master regulator of cellular energy homeostasis, and its activity depends on the AMP:ATP ratio. The cellular energy status and AMPK activation are connected by AMP, an allosteric activator of AMPK. Hence, an indirect strategy to affect AMPK activity would be to target the pathways that generate AMP in the cell. Herein, we report an up-to-date review of the interplay between AMPK and adenylate metabolizing enzymes. Some aspects of inborn errors of purine metabolism are also discussed. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

MIG1-dependent and MIG1-independent regulation of GAL gene expression in Saccharomyces cerevisiae: role of Imp2p.

PubMed

Alberti, Adriana; Lodi, Tiziana; Ferrero, Iliana; Donnini, Claudia

2003-10-15

Imp2p (Yil154c) is a transcriptional activator involved in glucose derepression of the maltose, galactose and raffinose utilization pathways and in resistance to thermal, oxidative or osmotic stress. We analysed the role of Imp2 in the regulation of GAL genes. Imp2 was shown to have a positive effect on glucose derepression of Leloir pathway genes and their activator gene GAL4. The effect of Imp2 on galactose metabolism was shown to be partially dependent on Mig1p. The Mig1-independent role depends on Nrg1p. However, disruption of both MIG1 and NRG1 only partially relieves the glucose repression of GAL genes in the Deltaimp2 mutant, indicating that Imp2 must also have other function(s). Moreover, the interaction between IMP2 and GAL6/BLH1, a recently isolated gene involved in the regulation of GAL genes that shares with Imp2 the ability to protect cells from the glycopeptide bleomycin, was also analysed. The results suggest a major role of Imp2 in a GAL6-independent pathway. Copyright 2003 John Wiley & Sons, Ltd.

p205, a potential tumor suppressor, inhibits cell proliferation via multiple pathways of cell cycle regulation.

PubMed

Asefa, Benyam; Dermott, Jonathan M; Kaldis, Philipp; Stefanisko, Karen; Garfinkel, David J; Keller, Jonathan R

2006-02-20

p205 is a member of the interferon-inducible p200 family of proteins that regulate cell proliferation. Over-expression of p205 inhibits cell growth, although its mechanism of action is currently unknown. Therefore, we evaluated the effect of p205 on the p53 and Rb-dependent pathways of cell cycle regulation. p205 expression results in elevated levels of p21, and activates the p21 promoter in vitro in a p53-dependent manner. In addition, p205 induces increased expression of Rb, and binds directly to Rb and p53. Interestingly, p205 also induces growth inhibition independent of p53 and Rb by delaying G2/M progression in proliferating cells, and is a substrate for Cdk2 kinase activity. Finally, we have identified other binding partners of p205 by a yeast two-hybrid screen, including the paired homeodomain protein HoxB2. Taken together, our results indicate that p205 induces growth arrest by interaction with multiple transcription factors that regulate the cell cycle, including but not entirely dependent on the Rb- and p53-mediated pathways of growth inhibition.

LPS-induced chemokine expression in both MyD88-dependent and -independent manners is regulated by Cot/Tpl2-ERK axis in macrophages.

PubMed

Bandow, Kenjiro; Kusuyama, Joji; Shamoto, Mitsuo; Kakimoto, Kyoko; Ohnishi, Tomokazu; Matsuguchi, Tetsuya

2012-05-21

LPS signaling is mediated through MyD88-dependent and -independent pathways, activating NF-?B, MAP kinases and IRF3. Cot/Tpl2 is an essential upstream kinase in LPS-mediated activation of ERKs. Here we explore the roles of MyD88 and Cot/Tpl2 in LPS-induced chemokine expression by studying myd88(-/-) and cot/tpl2(-/-) macrophages. Among the nine LPS-responsive chemokines examined, mRNA induction of ccl5, cxcl10, and cxcl13 is mediated through the MyD88-independent pathway. Notably, Cot/Tpl2-ERK signaling axis exerts negative effects on the expression of these three chemokines. In contrast, LPS-induced gene expression of ccl2, ccl7, cxcl2, cxcl3, ccl8, and cxcl9 is mediated in the MyD88-dependent manner. The Cot/Tpl2-ERK axis promotes the expression of the first four and inhibits the expression of the latter two. Thus, LPS induces expression of multiple chemokines through various signaling pathways in macrophages. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Anti-Inflammatory Action of an Antimicrobial Model Peptide That Suppresses the TRIF-Dependent Signaling Pathway via Inhibition of Toll-Like Receptor 4 Endocytosis in Lipopolysaccharide-Stimulated Macrophages

PubMed Central

Shim, Do-Wan; Heo, Kang-Hyuck; Kim, Young-Kyu; Sim, Eun-Jeong; Kang, Tae-Bong; Choi, Jae-Wan; Sim, Dae-Won; Cheong, Sun-Hee; Lee, Seung-Hong; Bang, Jeong-Kyu; Won, Hyung-Sik; Lee, Kwang-Ho

2015-01-01

Antimicrobial peptides (AMPs), also called host defense peptides, particularly those with amphipathic helical structures, are emerging as target molecules for therapeutic development due to their immunomodulatory properties. Although the antimicrobial activity of AMPs is known to be exerted primarily by permeation of the bacterial membrane, the mechanism underlying its anti-inflammatory activity remains to be elucidated. We report potent anti-inflammatory activity of WALK11.3, an antimicrobial model peptide with an amphipathic helical conformation, in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. This peptide inhibited the expression of inflammatory mediators, including nitric oxide, COX-2, IL-1β, IL-6, INF-β, and TNF-α. Although WALK11.3 did not exert a major effect on all downstream signaling in the MyD88-dependent pathway, toll-like receptor 4 (TLR4)- mediated pro-inflammatory signals were markedly attenuated in the TRIF-dependent pathway due to inhibition of the phosphorylation of STAT1 by attenuation of IRF3 phosphorylation. WALK11.3 specifically inhibited the endocytosis of TLR4, which is essential for triggering TRIF-mediated signaling in macrophage cells. Hence, we suggest that specific interference with TLR4 endocytosis could be one of the major modes of the anti-inflammatory action of AMPs. Our designed WALK11 peptides, which possess both antimicrobial and anti-inflammatory activities, may be promising molecules for the development of therapies for infectious inflammation. PMID:26017270

Anti-Inflammatory Action of an Antimicrobial Model Peptide That Suppresses the TRIF-Dependent Signaling Pathway via Inhibition of Toll-Like Receptor 4 Endocytosis in Lipopolysaccharide-Stimulated Macrophages.

PubMed

Shim, Do-Wan; Heo, Kang-Hyuck; Kim, Young-Kyu; Sim, Eun-Jeong; Kang, Tae-Bong; Choi, Jae-Wan; Sim, Dae-Won; Cheong, Sun-Hee; Lee, Seung-Hong; Bang, Jeong-Kyu; Won, Hyung-Sik; Lee, Kwang-Ho

2015-01-01

Antimicrobial peptides (AMPs), also called host defense peptides, particularly those with amphipathic helical structures, are emerging as target molecules for therapeutic development due to their immunomodulatory properties. Although the antimicrobial activity of AMPs is known to be exerted primarily by permeation of the bacterial membrane, the mechanism underlying its anti-inflammatory activity remains to be elucidated. We report potent anti-inflammatory activity of WALK11.3, an antimicrobial model peptide with an amphipathic helical conformation, in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. This peptide inhibited the expression of inflammatory mediators, including nitric oxide, COX-2, IL-1β, IL-6, INF-β, and TNF-α. Although WALK11.3 did not exert a major effect on all downstream signaling in the MyD88-dependent pathway, toll-like receptor 4 (TLR4)- mediated pro-inflammatory signals were markedly attenuated in the TRIF-dependent pathway due to inhibition of the phosphorylation of STAT1 by attenuation of IRF3 phosphorylation. WALK11.3 specifically inhibited the endocytosis of TLR4, which is essential for triggering TRIF-mediated signaling in macrophage cells. Hence, we suggest that specific interference with TLR4 endocytosis could be one of the major modes of the anti-inflammatory action of AMPs. Our designed WALK11 peptides, which possess both antimicrobial and anti-inflammatory activities, may be promising molecules for the development of therapies for infectious inflammation.

Balance between apoptosis or survival induced by changes in extracellular-matrix composition in human mesangial cells: a key role for ILK-NFκB pathway.

PubMed

del Nogal, María; Luengo, Alicia; Olmos, Gemma; Lasa, Marina; Rodriguez-Puyol, Diego; Rodriguez-Puyol, Manuel; Calleros, Laura

2012-12-01

Renal fibrosis is the final outcome of many clinical conditions that lead to chronic renal failure, characterized by a progressive substitution of cellular elements by extracellular-matrix proteins, in particular collagen type I. The aim of this study was to identify the mechanisms responsible for human mesangial cell survival, conditioned by changes in extracellular-matrix composition. Our results indicate that collagen I induces apoptosis in cells but only after inactivation of the pro-survival factor NFκB by either the super-repressor IκBα or the PDTC inhibitor. Collagen I activates a death pathway, through ILK/GSK-3β-dependent Bim expression. Moreover, collagen I significantly increases NFκB-dependent transcription, IκBα degradation and p65/NFκB translocation to the nucleus; it activates β1 integrin and this is accompanied by increased activity of ILK which leads to AKT activation. Knockdown of ILK or AKT with small interfering RNA suppresses the increase in NFκB activity. NFκB mediates cell survival through the antiapoptotic protein Bcl-xL. Our data suggest that human mesangial cells exposed to abnormal collagen I are protected against apoptosis by a complex mechanism involving integrin β1/ILK/AKT-dependent NFκB activation with consequent Bcl-xL overexpression, that opposes a simultaneously activated ILK/GSK-3β-dependent Bim expression and this dual mechanism may play a role in the progression of glomerular dysfunction.

Activity-dependent downregulation of M-Type (Kv7) K⁺ channels surface expression requires the activation of iGluRs/Ca²⁺/PKC signaling pathway in hippocampal neuron.

PubMed

Li, Cai; Lu, Qing; Huang, Pengcheng; Fu, Tianli; Li, Changjun; Guo, Lianjun; Xu, Xulin

2015-08-01

M-type (Kv7) K(+) channels, encoded by KCNQ2-KCNQ5 genes, play a pivotal role in controlling neuronal excitability. However, precisely how neuronal activity regulates Kv7 channel translocation has not yet been fully defined. Here we reported activity-dependent changes in Kv7 channel subunits Kv7.2 and Kv7.3 surface expression by glutamate (glu). In the present study, we found that treatment with glutamate rapidly caused a specific decrease in M-current as well as Kv7 channel surface expression in primary cultured hippocampal neurons. The glutamate effects were mimicked by NMDA and AMPA. The glutamate effects on Kv7 channels were partially attenuated by pre-treatment of NMDA receptors antagonist d,l-APV or AMPA-KA receptors antagonist CNQX. The signal required Ca(2+) influx through L-type Ca(2+) channel and intracellular Ca(2+) elevations. PKC activation was involved in the glutamate-induced reduction of Kv7 channel surface expression. Moreover, a significant reduction of Kv7 channel surface expression occurred following glycine-induced "chem"-LTP in vitro and hippocampus-dependent behavioral learning training in vivo. These results demonstrated that activity-dependent reduction of Kv7 channel surface expression through activation of ionotropic glutamate receptors (iGluRs)/Ca(2+)/PKC signaling pathway might be an important molecular mechanism for regulation of neuronal excitability and synaptic plasticity. Copyright © 2015 Elsevier Ltd. All rights reserved.

Benchmarking pathway interaction network for colorectal cancer to identify dysregulated pathways.

PubMed

Wang, Q; Shi, C-J; Lv, S-H

2017-03-30

Different pathways act synergistically to participate in many biological processes. Thus, the purpose of our study was to extract dysregulated pathways to investigate the pathogenesis of colorectal cancer (CRC) based on the functional dependency among pathways. Protein-protein interaction (PPI) information and pathway data were retrieved from STRING and Reactome databases, respectively. After genes were aligned to the pathways, each pathway activity was calculated using the principal component analysis (PCA) method, and the seed pathway was discovered. Subsequently, we constructed the pathway interaction network (PIN), where each node represented a biological pathway based on gene expression profile, PPI data, as well as pathways. Dysregulated pathways were then selected from the PIN according to classification performance and seed pathway. A PIN including 11,960 interactions was constructed to identify dysregulated pathways. Interestingly, the interaction of mRNA splicing and mRNA splicing-major pathway had the highest score of 719.8167. Maximum change of the activity score between CRC and normal samples appeared in the pathway of DNA replication, which was selected as the seed pathway. Starting with this seed pathway, a pathway set containing 30 dysregulated pathways was obtained with an area under the curve score of 0.8598. The pathway of mRNA splicing, mRNA splicing-major pathway, and RNA polymerase I had the maximum genes of 107. Moreover, we found that these 30 pathways had crosstalks with each other. The results suggest that these dysregulated pathways might be used as biomarkers to diagnose CRC.

Tumor-propagating cells and Yap/Taz activity contribute to lung tumor progression and metastasis

PubMed Central

Lau, Allison N; Curtis, Stephen J; Fillmore, Christine M; Rowbotham, Samuel P; Mohseni, Morvarid; Wagner, Darcy E; Beede, Alexander M; Montoro, Daniel T; Sinkevicius, Kerstin W; Walton, Zandra E; Barrios, Juliana; Weiss, Daniel J; Camargo, Fernando D; Wong, Kwok-Kin; Kim, Carla F

2014-01-01

Metastasis is the leading cause of morbidity for lung cancer patients. Here we demonstrate that murine tumor propagating cells (TPCs) with the markers Sca1 and CD24 are enriched for metastatic potential in orthotopic transplantation assays. CD24 knockdown decreased the metastatic potential of lung cancer cell lines resembling TPCs. In lung cancer patient data sets, metastatic spread and patient survival could be stratified with a murine lung TPC gene signature. The TPC signature was enriched for genes in the Hippo signaling pathway. Knockdown of the Hippo mediators Yap1 or Taz decreased in vitro cellular migration and transplantation of metastatic disease. Furthermore, constitutively active Yap was sufficient to drive lung tumor progression in vivo. These results demonstrate functional roles for two different pathways, CD24-dependent and Yap/Taz-dependent pathways, in lung tumor propagation and metastasis. This study demonstrates the utility of TPCs for identifying molecules contributing to metastatic lung cancer, potentially enabling the therapeutic targeting of this devastating disease. PMID:24497554

Agaricus blazei Extract Induces Apoptosis through ROS-Dependent JNK Activation Involving the Mitochondrial Pathway and Suppression of Constitutive NF-κB in THP-1 Cells

PubMed Central

Kim, Mun-Ock; Moon, Dong-Oh; Jung, Jin Myung; Lee, Won Sup; Choi, Yung Hyun; Kim, Gi-Young

2011-01-01

Agaricus blazei is widely accepted as a traditional medicinal mushroom, and it has been known to exhibit immunostimulatory and anti-cancer activity. However, the apoptotic mechanism in cancer cells is poorly understood. In this study, we have investigated whether A. blazei extract (ABE) exerts antiproliferative and apoptotic effects in human leukemic THP-1 cells. We observed that ABE-induced apoptosis is associated with the mitochondrial pathway, which is mediated by reactive oxygen species (ROS) generation and prolonged c-Jun N-terminal kinase (JNK) activation. In addition, the ABE treatment resulted in the accumulation of cytochrome c in the cytoplasm, an increase in caspase activity, and an upregulation of Bax and Bad. With those results in mind, we found that ABE decreases constitutive NF-κB activation and NF-κB-regulated gene products such as IAP-1 and -2. We concluded that ABE induces apoptosis with ROS-dependent JNK activation and constitutive activated NF-κB inhibition in THP-1 cells. PMID:19861509

Induction of Direct Antimicrobial Activity Through Mammalian Toll-Like Receptors

NASA Astrophysics Data System (ADS)

Thoma-Uszynski, Sybille; Stenger, Steffen; Takeuchi, Osamu; Ochoa, Maria Teresa; Engele, Matthias; Sieling, Peter A.; Barnes, Peter F.; Röllinghoff, Martin; Bölcskei, Pal L.; Wagner, Manfred; Akira, Shizuo; Norgard, Michael V.; Belisle, John T.; Godowski, Paul J.; Bloom, Barry R.; Modlin, Robert L.

2001-02-01

The mammalian innate immune system retains from Drosophila a family of homologous Toll-like receptors (TLRs) that mediate responses to microbial ligands. Here, we show that TLR2 activation leads to killing of intracellular Mycobacterium tuberculosis in both mouse and human macrophages, through distinct mechanisms. In mouse macrophages, bacterial lipoprotein activation of TLR2 leads to a nitric oxide-dependent killing of intracellular tubercle bacilli, but in human monocytes and alveolar macrophages, this pathway was nitric oxide-independent. Thus, mammalian TLRs respond (as Drosophila Toll receptors do) to microbial ligands and also have the ability to activate antimicrobial effector pathways at the site of infection.

Arachidonic acid stimulates DNA synthesis in brown preadipocytes through the activation of protein kinase C and MAPK.

PubMed

Garcia, Bibian; Martinez-de-Mena, Raquel; Obregon, Maria-Jesus

2012-10-01

Arachidonic acid (AA) is a polyunsaturated fatty acid that stimulates the proliferation of many cellular types. We studied the mitogenic potential of AA in rat brown preadipocytes in culture and the signaling pathways involved. AA is a potent mitogen which induces 4-fold DNA synthesis in brown preadipocytes. The AA mitogenic effect increases by NE addition. AA also increases the mitogenic action of different growth factor combinations. Other unsaturated and saturated fatty acids do not stimulate DNA synthesis to the same extent as AA. We analyzed the role of PKC and MEK/MAPK signaling pathways. PKC inhibition by bisindolilmaleimide I (BIS) abolishes AA and phorbol ester stimulation of DNA synthesis and reduces the mitogenic activity of different growth factors in brown preadipocytes. Brown preadipocytes in culture express PKC α, δ, ε and ζ isoforms. Pretreatment with high doses of the phorbol ester PDBu, induces downregulation of PKCs ε and δ and reproduces the effect of BIS indicating that AA-dependent induction of DNA synthesis requires PKC activity. AA also activates MEK/MAPK pathway and the inhibition of MEK activity inhibits AA stimulation of DNA synthesis and brown adipocyte proliferation. Inhibition of PKC δ by rottlerin abolishes AA-dependent stimulation of DNA synthesis and MAPK activation, whereas PKC ε inhibition does not produce any effect. In conclusion, our results identify AA as a potent mitogen for brown adipocytes and demonstrate the involvement of the PDBu-sensitive PKC δ isoform and MEK/MAPK pathway in AA-induced proliferation of brown adipocytes. Increased proliferative activity might increase the thermogenic capacity of brown fat. Copyright © 2012 Elsevier B.V. All rights reserved.

XEDAR activates the non-canonical NF-κB pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Verhelst, Kelly, E-mail: Kelly.Verhelst@irc.VIB-UGent.be; Department of Biomedical Molecular Biology, Ghent University, Ghent; Gardam, Sandra, E-mail: s.gardam@garvan.org.au

2015-09-18

Members of the tumor necrosis factor receptor (TNFR) superfamily are involved in a number of physiological and pathological responses by activating a wide variety of intracellular signaling pathways. The X-linked ectodermal dysplasia receptor (XEDAR; also known as EDA2R or TNFRSF27) is a member of the TNFR superfamily that is highly expressed in ectodermal derivatives during embryonic development and binds to ectodysplasin-A2 (EDA-A2), a member of the TNF family that is encoded by the anhidrotic ectodermal dysplasia (EDA) gene. Although XEDAR was first described in the year 2000, its function and molecular mechanism of action is still largely unclear. XEDAR hasmore » been reported to activate canonical nuclear factor κB (NF-κB) signaling and mitogen-activated protein (MAP) kinases. Here we report that XEDAR is also able to trigger the non-canonical NF-κB pathway, characterized by the processing of p100 (NF-κB2) into p52, followed by nuclear translocation of p52 and RelB. We provide evidence that XEDAR-induced p100 processing relies on the binding of XEDAR to TRAF3 and TRAF6, and requires the kinase activity of NIK and IKKα. We also show that XEDAR stimulation results in NIK accumulation and that p100 processing is negatively regulated by TRAF3, cIAP1 and A20. - Highlights: • XEDAR activates the non-canonical NF-κB pathway. • XEDAR-induced processing of p100 depends on XEDAR interaction with TRAF3 and TRAF6. • XEDAR-induced processing of p100 depends on NIK and IKKα activity. • Overexpression of XEDAR leads to NIK accumulation. • XEDAR-induced processing of p100 is negatively regulated by TRAF3 cIAP1 and A20.« less

Pterostilbene protects against UVB-induced photo-damage through a phosphatidylinositol-3-kinase-dependent Nrf2/ARE pathway in human keratinocytes.

PubMed

Li, Huaping; Jiang, Na; Liang, Bihua; Liu, Qing; Zhang, Erting; Peng, Liqian; Deng, Huiyan; Li, Runxiang; Li, Zhenjie; Zhu, Huilan

2017-11-01

Ultraviolet B (UVB) irradiation is the initial etiological factor for various skin disorders, including erythema, sunburn, photoaging, and photocarcinogenesis. Pterostilbene (Pter) displayed remarkable antioxidant, anti-inflammatory, and anticarcinogenic activities. This study aimed to investigate the effective mechanism of Pter against UVB-induced photodamage in immortalized human keratinocytes. Human keratinocytes were pretreated with Pter (5 and 10 μM) for 24 h prior to UVB irradiation (300 mJ/cm 2 ). Harvested cells were analyzed by MTT, DCFH-DA, comet, western blotting, luciferase promoter, small interference RNA transfection, and quantitative real-time polymerase chain reaction assay. Pter significantly attenuated UVB-induced cell death and reactive oxygen species (ROS) generation, and effectively increased nuclear translocation of NF-E2-related factor-2 (Nrf2), expression of Nrf2-dependent antioxidant enzymes, and DNA repair activity. Moreover, the protective effects of Pter were abolished by small interference RNA-mediated Nrf2 silencing. Furthermore, Pter was also found to induce the phosphorylation of Nrf2 and the known phosphatidylinositol-3-kinase (PI3K) phosphorylated kinase, Akt. The specific inhibitor of PI3K, LY294002, successfully abrogated Pter-induced Nrf2 phosphorylation, activation of Nrf2-antioxidant response element pathway, ROS scavenging ability, and DNA repair activity. The present study indicated that Pter effectively protected against UVB-induced photodamage by increasing endogenous defense mechanisms, scavenging UVB-induced ROS, and aiding in damaged DNA repair through a PI3K-dependent activation of Nrf2/ARE pathway.

Cyclin-dependent kinase 5-mediated phosphorylation of CHIP promotes the tAIF-dependent death pathway in rotenone-treated cortical neurons.

PubMed

Kim, Chiho; Lee, Juhyung; Ko, Yeon Uk; Oh, Young J

2018-01-01

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase. Its dysregulation has been implicated in various neurodegenerative diseases. We previously reported that phosphorylation of the C-terminus of the Hsc70-interacting protein (CHIP) by Cdk5 promotes truncated apoptosis-inducing factor (tAIF)-mediated neuronal death induced by oxidative stress. Here, we determined whether this Cdk5-dependent cell death signaling pathway is present in experimental models of Parkinson's disease. First, we showed that rotenone activates Cdk5 in primary cultures of cortical neurons and causes tAIF-dependent neuronal cell death. This event was attenuated by negative regulation of endogenous Cdk5 activity by the pharmacological Cdk5 inhibitor, roscovitine, or by lentiviral knockdown of Cdk5. Cdk5 phosphorylates CHIP at Ser20 in rotenone-treated neurons. Consequently, overexpression of CHIP S20A , but not CHIP WT , attenuates tAIF-induced cell death in rotenone-treated cortical neurons. Taken together, these results indicate that phosphorylation of CHIP at Ser20 by Cdk5 activation inhibits CHIP-mediated tAIF degradation, thereby contributing to tAIF-induced neuronal cell death following rotenone treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

Protein kinase C and P2Y12 take center stage in thrombin-mediated activation of mammalian target of rapamycin complex 1 in human platelets.

PubMed

Moore, S F; Hunter, R W; Hers, I

2014-05-01

Rapamycin, an inhibitor of mammalian target of rapamycin complex-1 (mTORC1), reduces platelet spreading, thrombus stability, and clot retraction. Despite an important role of mTORC1 in platelet function, little is known about how it is regulated. The objective of this study was to determine the signaling pathways that regulate mTORC1 in human platelets. Mammalian target of rapamycin complex-1 activation was assessed by measuring the phosphorylation of its downstream substrate ribosomal S6 kinase 1 (p70S6K). Thrombin or the protein kinase C (PKC) activator phorbal 12-myristate 13-acetate stimulated activation of mTORC1 in a PKC-dependent, Akt-independent manner that correlated with phosphorylation of tuberin/tuberous sclerosis 2 (TSC2) (Ser939 and Thr1462). In contrast, insulin-like growth factor 1 (IGF-1)-stimulated TSC2 phosphorylation was completely dependent on phosphoinositide 3 kinase (PI3 kinase)/Akt but did not result in any detectable mTORC1 activation. Early (Ser939 and Thr1462) and late (Thr1462) TSC2 phosphorylation in response to thrombin were directly PKC dependent, whereas later TSC2 (Ser939) and p70S6K phosphorylation were largely dependent on paracrine signaling through P2Y(12). PKC-mediated adenosine diphosphate (ADP) secretion was essential for thrombin-stimulated mTORC1 activation, as (i) ADP rescued p70S6K phosphorylation in the presence of a PKC inhibitor and (ii) P2Y(12) antagonism prevented thrombin-mediated mTORC1 activation. Rescue of mTORC1 activation with exogenous ADP was completely dependent on the Src family kinases but independent of PI3 kinase/Akt. Interestingly, although inhibition of Src blocked the ADP rescue, it had little effect on thrombin-stimulated p70S6K phosphorylation under conditions where PKC was not inhibited. These results demonstrate that thrombin activates the mTORC1 pathway in human platelets through PKC-mediated ADP secretion and subsequent activation of P2Y(12), in a manner largely independent of the canonical PI3 kinase/Akt pathway. © 2014 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.

Protein kinase C and P2Y12 take center stage in thrombin-mediated activation of mammalian target of rapamycin complex 1 in human platelets

PubMed Central

Moore, S F; Hunter, R W; Hers, I

2014-01-01

Background Rapamycin, an inhibitor of mammalian target of rapamycin complex-1 (mTORC1), reduces platelet spreading, thrombus stability, and clot retraction. Despite an important role of mTORC1 in platelet function, little is known about how it is regulated. The objective of this study was to determine the signaling pathways that regulate mTORC1 in human platelets. Methods Mammalian target of rapamycin complex-1 activation was assessed by measuring the phosphorylation of its downstream substrate ribosomal S6 kinase 1 (p70S6K). Results Thrombin or the protein kinase C (PKC) activator phorbal 12-myristate 13-acetate stimulated activation of mTORC1 in a PKC-dependent, Akt-independent manner that correlated with phosphorylation of tuberin/tuberous sclerosis 2 (TSC2) (Ser939 and Thr1462). In contrast, insulin-like growth factor 1 (IGF-1)–stimulated TSC2 phosphorylation was completely dependent on phosphoinositide 3 kinase (PI3 kinase)/Akt but did not result in any detectable mTORC1 activation. Early (Ser939 and Thr1462) and late (Thr1462) TSC2 phosphorylation in response to thrombin were directly PKC dependent, whereas later TSC2 (Ser939) and p70S6K phosphorylation were largely dependent on paracrine signaling through P2Y12. PKC-mediated adenosine diphosphate (ADP) secretion was essential for thrombin-stimulated mTORC1 activation, as (i) ADP rescued p70S6K phosphorylation in the presence of a PKC inhibitor and (ii) P2Y12 antagonism prevented thrombin-mediated mTORC1 activation. Rescue of mTORC1 activation with exogenous ADP was completely dependent on the Src family kinases but independent of PI3 kinase/Akt. Interestingly, although inhibition of Src blocked the ADP rescue, it had little effect on thrombin-stimulated p70S6K phosphorylation under conditions where PKC was not inhibited. Conclusion These results demonstrate that thrombin activates the mTORC1 pathway in human platelets through PKC-mediated ADP secretion and subsequent activation of P2Y12, in a manner largely independent of the canonical PI3 kinase/Akt pathway. PMID:24612393

Experimental hyperthyroidism in rats increases the expression of the ubiquitin ligases atrogin-1 and MuRF1 and stimulates multiple proteolytic pathways in skeletal muscle.

PubMed

O'Neal, Patrick; Alamdari, Nima; Smith, Ira; Poylin, Vitaliy; Menconi, Michael; Hasselgren, Per-Olof

2009-11-01

Muscle wasting is commonly seen in patients with hyperthyroidism and is mainly caused by stimulated muscle proteolysis. Loss of muscle mass in several catabolic conditions is associated with increased expression of the muscle-specific ubiquitin ligases atrogin-1 and MuRF1 but it is not known if atrogin-1 and MuRF1 are upregulated in hyperthyroidism. In addition, it is not known if thyroid hormone increases the activity of proteolytic mechanisms other than the ubiquitin-proteasome pathway. We tested the hypotheses that experimental hyperthyroidism in rats, induced by daily intraperitoneal injections of 100 microg/100 g body weight of triiodothyronine (T3), upregulates the expression of atrogin-1 and MuRF1 in skeletal muscle and stimulates lysosomal, including cathepsin L, calpain-, and caspase-3-dependent protein breakdown in addition to proteasome-dependent protein breakdown. Treatment of rats with T3 for 3 days resulted in an approximately twofold increase in atrogin-1 and MuRF1 mRNA levels. The same treatment increased proteasome-, cathepsin L-, and calpain-dependent proteolytic rates by approximately 40% but did not influence caspase-3-dependent proteolysis. The expression of atrogin-1 and MuRF1 remained elevated during a more prolonged period (7 days) of T3 treatment. The results provide support for a role of the ubiquitin-proteasome pathway in muscle wasting during hyperthyroidism and suggest that other proteolytic pathways as well may be activated in the hyperthyroid state. (c) 2009 Wiley-Liss, Inc.

SARS coronavirus papain-like protease induces Egr-1-dependent up-regulation of TGF-β1 via ROS/p38 MAPK/STAT3 pathway

PubMed Central

Li, Shih-Wein; Wang, Ching-Ying; Jou, Yu-Jen; Yang, Tsuey-Ching; Huang, Su-Hua; Wan, Lei; Lin, Ying-Ju; Lin, Cheng-Wen

2016-01-01

SARS coronavirus (SARS-CoV) papain-like protease (PLpro) has been identified in TGF-β1 up-regulation in human promonocytes (Proteomics 2012, 12: 3193-205). This study investigates the mechanisms of SARS-CoV PLpro-induced TGF-β1 promoter activation in human lung epithelial cells and mouse models. SARS-CoV PLpro dose- and time-dependently up-regulates TGF-β1 and vimentin in A549 cells. Dual luciferase reporter assays with TGF-β1 promoter plasmids indicated that TGF-β1 promoter region between −175 to −60, the Egr-1 binding site, was responsible for TGF-β1 promoter activation induced by SARS-CoV PLpro. Subcellular localization analysis of transcription factors showed PLpro triggering nuclear translocation of Egr-1, but not NF-κB and Sp-1. Meanwhile, Egr-1 silencing by siRNA significantly reduced PLpro-induced up-regulation of TGF-β1, TSP-1 and pro-fibrotic genes. Furthermore, the inhibitors for ROS (YCG063), p38 MAPK (SB203580), and STAT3 (Stattic) revealed ROS/p38 MAPK/STAT3 pathway involving in Egr-1 dependent activation of TGF-β1 promoter induced by PLpro. In a mouse model with a direct pulmonary injection, PLpro stimulated macrophage infiltration into lung, up-regulating Egr-1, TSP-1, TGF-β1 and vimentin expression in lung tissues. The results revealed that SARS-CoV PLpro significantly triggered Egr-1 dependent activation of TGF-β1 promoter via ROS/p38 MAPK/STAT3 pathway, correlating with up-regulation of pro-fibrotic responses in vitro and in vivo. PMID:27173006

SARS coronavirus papain-like protease induces Egr-1-dependent up-regulation of TGF-β1 via ROS/p38 MAPK/STAT3 pathway.

PubMed

Li, Shih-Wein; Wang, Ching-Ying; Jou, Yu-Jen; Yang, Tsuey-Ching; Huang, Su-Hua; Wan, Lei; Lin, Ying-Ju; Lin, Cheng-Wen

2016-05-13

SARS coronavirus (SARS-CoV) papain-like protease (PLpro) has been identified in TGF-β1 up-regulation in human promonocytes (Proteomics 2012, 12: 3193-205). This study investigates the mechanisms of SARS-CoV PLpro-induced TGF-β1 promoter activation in human lung epithelial cells and mouse models. SARS-CoV PLpro dose- and time-dependently up-regulates TGF-β1 and vimentin in A549 cells. Dual luciferase reporter assays with TGF-β1 promoter plasmids indicated that TGF-β1 promoter region between -175 to -60, the Egr-1 binding site, was responsible for TGF-β1 promoter activation induced by SARS-CoV PLpro. Subcellular localization analysis of transcription factors showed PLpro triggering nuclear translocation of Egr-1, but not NF-κB and Sp-1. Meanwhile, Egr-1 silencing by siRNA significantly reduced PLpro-induced up-regulation of TGF-β1, TSP-1 and pro-fibrotic genes. Furthermore, the inhibitors for ROS (YCG063), p38 MAPK (SB203580), and STAT3 (Stattic) revealed ROS/p38 MAPK/STAT3 pathway involving in Egr-1 dependent activation of TGF-β1 promoter induced by PLpro. In a mouse model with a direct pulmonary injection, PLpro stimulated macrophage infiltration into lung, up-regulating Egr-1, TSP-1, TGF-β1 and vimentin expression in lung tissues. The results revealed that SARS-CoV PLpro significantly triggered Egr-1 dependent activation of TGF-β1 promoter via ROS/p38 MAPK/STAT3 pathway, correlating with up-regulation of pro-fibrotic responses in vitro and in vivo.

Bidirectional reporter assay using HAL promoter and TOPFLASH improves specificity in high-throughput screening of Wnt inhibitors.

PubMed

Yamaguchi, Kiyoshi; Zhu, Chi; Ohsugi, Tomoyuki; Yamaguchi, Yuko; Ikenoue, Tsuneo; Furukawa, Yoichi

2017-12-01

Constitutive activation of Wnt signaling plays an important role in colorectal and liver tumorigenesis. Cell-based assays using synthetic TCF/LEF (T-cell factor/lymphoid enhancer factor) reporters, as readouts of β-catenin/TCF-dependent transcriptional activity, have contributed greatly to the discovery of small molecules that modulate Wnt signaling. In the present study, we report a novel screening method, called a bidirectional dual reporter assay. Integrated transcriptome analysis identified a histidine ammonia-lyase gene (HAL) that was negatively regulated by β-catenin/TCF-dependent transcriptional activity. We leveraged a promoter region of the HAL gene as another transcriptional readout of Wnt signaling. Cells stably expressing both an optimized HAL reporter and the TCF/LEF reporter enabled bidirectional reporter activities in response to Wnt signaling. Increased HAL reporter activity and decreased TCF/LEF reporter activity were observed simultaneously in the cells when β-catenin/TCF7L2 was inhibited. Notably, this method could decrease the number of false positives observed when screening an inhibitor library compared with the conventional TCF/LEF assay. We found that Brefeldin A, a disruptor of the Golgi apparatus, inhibited the Wnt/β-catenin signaling pathway. The utility of our system could be expanded to examine other disease-associated pathways beyond the Wnt/β-catenin signaling pathway. © 2017 Wiley Periodicals, Inc.

Phosphodiesterase inhibitors suppress Lactobacillus casei cell-wall-induced NF-κB and MAPK activations and cell proliferation through protein kinase A--or exchange protein activated by cAMP-dependent signal pathway.

PubMed

Saito, Takekatsu; Sugimoto, Naotoshi; Ohta, Kunio; Shimizu, Tohru; Ohtani, Kaori; Nakayama, Yuko; Nakamura, Taichi; Hitomi, Yashiaki; Nakamura, Hiroyuki; Koizumi, Shoichi; Yachie, Akihiro

2012-01-01

Specific strains of Lactobacillus have been found to be beneficial in treating some types of diarrhea and vaginosis. However, a high mortality rate results from underlying immunosuppressive conditions in patients with Lactobacillus casei bacteremia. Cyclic AMP (cAMP) is a small second messenger molecule that mediates signal transduction. The onset and progression of inflammatory responses are sensitive to changes in steady-state cAMP levels. L. casei cell wall extract (LCWE) develops arteritis in mice through Toll-like receptor-2 signaling. The purpose of this study was to investigate whether intracellular cAMP affects LCWE-induced pathological signaling. LCWE was shown to induce phosphorylation of the nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways and cell proliferation in mice fibroblast cells. Theophylline and phosphodiesterase inhibitor increased intracellular cAMP and inhibited LCWE-induced cell proliferation as well as phosphorylation of NF-κB and MAPK. Protein kinase A inhibitor H89 prevented cAMP-induced MAPK inhibition, but not cAMP-induced NF-κB inhibition. An exchange protein activated by cAMP (Epac) agonist inhibited NF-κB activation but not MAPK activation. These results indicate that an increase in intracellular cAMP prevents LCWE induction of pathological signaling pathways dependent on PKA and Epac signaling.

Age-dependent regulation of ERF-VII transcription factor activity in Arabidopsis thaliana.

PubMed

Giuntoli, Beatrice; Shukla, Vinay; Maggiorelli, Federica; Giorgi, Federico M; Lombardi, Lara; Perata, Pierdomenico; Licausi, Francesco

2017-10-01

The Group VII Ethylene Responsive Factors (ERFs-VII) RAP2.2 and RAP2.12 have been mainly characterized with regard to their contribution as activators of fermentation in plants. However, transcriptional changes measured in conditions that stabilize these transcription factors exceed the mere activation of this biochemical pathway, implying additional roles performed by the ERF-VIIs in other processes. We evaluated gene expression in transgenic Arabidopsis lines expressing a stabilized form of RAP2.12, or hampered in ERF-VII activity, and identified genes affected by this transcriptional regulator and its homologs, including some involved in oxidative stress response, which are not universally induced under anaerobic conditions. The contribution of the ERF-VIIs in regulating this set of genes in response to chemically induced or submergence-stimulated mitochondria malfunctioning was found to depend on the plant developmental stage. A similar age-dependent mechanism also restrained ERF-VII activity upon the core-hypoxic genes, independently of the N-end rule pathway, which is accounted for the control of the anaerobic response. To conclude, this study shed new light on a dual role of ERF-VII proteins under submergence: as positive regulators of the hypoxic response and as repressors of oxidative-stress related genes, depending on the developmental stage at which plants are challenged by stress conditions. © 2017 John Wiley & Sons Ltd.

PTP1B triggers integrin-mediated repression of myosin activity and modulates cell contractility

PubMed Central

González Wusener, Ana E.; González, Ángela; Nakamura, Fumihiko; Arregui, Carlos O.

2016-01-01

ABSTRACT Cell contractility and migration by integrins depends on precise regulation of protein tyrosine kinase and Rho-family GTPase activities in specific spatiotemporal patterns. Here we show that protein tyrosine phosphatase PTP1B cooperates with β3 integrin to activate the Src/FAK signalling pathway which represses RhoA-myosin-dependent contractility. Using PTP1B null (KO) cells and PTP1B reconstituted (WT) cells, we determined that some early steps following cell adhesion to fibronectin and vitronectin occurred robustly in WT cells, including aggregation of β3 integrins and adaptor proteins, and activation of Src/FAK-dependent signalling at small puncta in a lamellipodium. However, these events were significantly impaired in KO cells. We established that cytoskeletal strain and cell contractility was highly enhanced at the periphery of KO cells compared to WT cells. Inhibition of the Src/FAK signalling pathway or expression of constitutive active RhoA in WT cells induced a KO cell phenotype. Conversely, expression of constitutive active Src or myosin inhibition in KO cells restored the WT phenotype. We propose that this novel function of PTP1B stimulates permissive conditions for adhesion and lamellipodium assembly at the protruding edge during cell spreading and migration. PMID:26700725

Isonicotinamide Enhances Sir2 Protein-mediated Silencing and Longevity in Yeast by Raising Intracellular NAD+ Concentration*

PubMed Central

McClure, Julie M.; Wierman, Margaret B.; Maqani, Nazif; Smith, Jeffrey S.

2012-01-01

Sirtuins are an evolutionarily conserved family of NAD+-dependent protein deacetylases that function in the regulation of gene transcription, cellular metabolism, and aging. Their activity requires the maintenance of an adequate intracellular NAD+ concentration through the combined action of NAD+ biosynthesis and salvage pathways. Nicotinamide (NAM) is a key NAD+ precursor that is also a byproduct and feedback inhibitor of the deacetylation reaction. In Saccharomyces cerevisiae, the nicotinamidase Pnc1 converts NAM to nicotinic acid (NA), which is then used as a substrate by the NAD+ salvage pathway enzyme NA phosphoribosyltransferase (Npt1). Isonicotinamide (INAM) is an isostere of NAM that stimulates yeast Sir2 deacetylase activity in vitro by alleviating the NAM inhibition. In this study, we determined that INAM stimulates Sir2 through an additional mechanism in vivo, which involves elevation of the intracellular NAD+ concentration. INAM enhanced normal silencing at the rDNA locus but only partially suppressed the silencing defects of an npt1Δ mutant. Yeast cells grown in media lacking NA had a short replicative life span, which was extended by INAM in a SIR2-dependent manner and correlated with increased NAD+. The INAM-induced increase in NAD+ was strongly dependent on Pnc1 and Npt1, suggesting that INAM increases flux through the NAD+ salvage pathway. Part of this effect was mediated by the NR salvage pathways, which generate NAM as a product and require Pnc1 to produce NAD+. We also provide evidence suggesting that INAM influences the expression of multiple NAD+ biosynthesis and salvage pathways to promote homeostasis during stationary phase. PMID:22539348

The nitric-oxide reductase from Paracoccus denitrificans uses a single specific proton pathway.

PubMed

ter Beek, Josy; Krause, Nils; Reimann, Joachim; Lachmann, Peter; Ädelroth, Pia

2013-10-18

The NO reductase from Paracoccus denitrificans reduces NO to N2O (2NO + 2H(+) + 2e(-) → N2O + H2O) with electrons donated by periplasmic cytochrome c (cytochrome c-dependent NO reductase; cNOR). cNORs are members of the heme-copper oxidase superfamily of integral membrane proteins, comprising the O2-reducing, proton-pumping respiratory enzymes. In contrast, although NO reduction is as exergonic as O2 reduction, there are no protons pumped in cNOR, and in addition, protons needed for NO reduction are derived from the periplasmic solution (no contribution to the electrochemical gradient is made). cNOR thus only needs to transport protons from the periplasm into the active site without the requirement to control the timing of opening and closing (gating) of proton pathways as is needed in a proton pump. Based on the crystal structure of a closely related cNOR and molecular dynamics simulations, several proton transfer pathways were suggested, and in principle, these could all be functional. In this work, we show that residues in one of the suggested pathways (denoted pathway 1) are sensitive to site-directed mutation, whereas residues in the other proposed pathways (pathways 2 and 3) could be exchanged without severe effects on turnover activity with either NO or O2. We further show that electron transfer during single-turnover reduction of O2 is limited by proton transfer and can thus be used to study alterations in proton transfer rates. The exchange of residues along pathway 1 showed specific slowing of this proton-coupled electron transfer as well as changes in its pH dependence. Our results indicate that only pathway 1 is used to transfer protons in cNOR.

Subsurface flow pathway dynamics in the active layer of coupled permafrost-hydrogeological systems under seasonal and annual temperature variability.

NASA Astrophysics Data System (ADS)

Frampton, Andrew

2017-04-01

There is a need for improved understanding of the mechanisms controlling subsurface solute transport in the active layer in order to better understand permafrost-hydrological-carbon feedbacks, in particular with regards to how dissolved carbon is transported in coupled surface and subsurface terrestrial arctic water systems under climate change. Studying solute transport in arctic systems is also relevant in the context of anthropogenic pollution which may increase due to increased activity in cold region environments. In this contribution subsurface solute transport subject to ground surface warming causing permafrost thaw and active layer change is studied using a physically based model of coupled cryotic and hydrogeological flow processes combined with a particle tracking method. Changes in subsurface water flows and solute transport travel times are analysed for different modelled geological configurations during a 100-year warming period. Results show that for all simulated cases, the minimum and mean travel times increase non-linearly with warming irrespective of geological configuration and heterogeneity structure. The timing of the start of increase in travel time depends on heterogeneity structure, combined with the rate of permafrost degradation that also depends on material thermal and hydrogeological properties. These travel time changes are shown to depend on combined warming effects of increase in pathway length due to deepening of the active layer, reduced transport velocities due to a shift from horizontal saturated groundwater flow near the surface to vertical water percolation deeper into the subsurface, and pathway length increase and temporary immobilization caused by cryosuction-induced seasonal freeze cycles. The impact these change mechanisms have on solute and dissolved substance transport is further analysed by integrating pathway analysis with a Lagrangian approach, incorporating considerations for both dissolved organic and inorganic carbon releases. Further model development challenges are also highlighted and discussed, including coupling between subsurface and surface runoff, soil deformations, as well as site applications and larger system scales.

Mechanistic Target of Rapamycin Complex 1 (mTORC1) and mTORC2 as Key Signaling Intermediates in Mesenchymal Cell Activation*

PubMed Central

Walker, Natalie M.; Belloli, Elizabeth A.; Stuckey, Linda; Chan, Kevin M.; Lin, Jules; Lynch, William; Chang, Andrew; Mazzoni, Serina M.; Fingar, Diane C.; Lama, Vibha N.

2016-01-01

Fibrotic diseases display mesenchymal cell (MC) activation with pathologic deposition of matrix proteins such as collagen. Here we investigate the role of mTOR complex 1 (mTORC1) and mTORC2 in regulating MC collagen expression, a hallmark of fibrotic disease. Relative to normal MCs (non-Fib MCs), MCs derived from fibrotic human lung allografts (Fib-MCs) demonstrated increased phosphoinositide-3kinase (PI3K) dependent activation of both mTORC1 and mTORC2, as measured by increased phosphorylation of S6K1 and 4E-BP1 (mTORC1 substrates) and AKT (an mTORC2 substrate). Dual ATP-competitive TORC1/2 inhibitor AZD8055, in contrast to allosteric mTORC1-specific inhibitor rapamycin, strongly inhibited 4E-BP1 phosphorylation and collagen I expression in Fib-MCs. In non-Fib MCs, increased mTORC1 signaling was shown to augment collagen I expression. mTORC1/4E-BP1 pathway was identified as an important driver of collagen I expression in Fib-MCs in experiments utilizing raptor gene silencing and overexpression of dominant-inhibitory 4E-BP1. Furthermore, siRNA-mediated knockdown of rictor, an mTORC2 partner protein, reduced mTORC1 substrate phosphorylation and collagen expression in Fib-, but not non-Fib MCs, revealing a dependence of mTORC1 signaling on mTORC2 function in activated MCs. Together these studies suggest a novel paradigm where fibrotic activation in MCs increases PI3K dependent mTORC1 and mTORC2 signaling and leads to increased collagen I expression via the mTORC1-dependent 4E-BP1/eIF4E pathway. These data provide rationale for targeting specific components of mTORC pathways in fibrotic states and underscore the need to further delineate mTORC2 signaling in activated cell states. PMID:26755732

Cross talk between the TM4SF5/focal adhesion kinase and the interleukin-6/STAT3 pathways promotes immune escape of human liver cancer cells.

PubMed

Ryu, Jihye; Kang, Minkyung; Lee, Mi-Sook; Kim, Hye-Jin; Nam, Seo Hee; Song, Haeng Eun; Lee, Doohyung; Lee, Jung Weon

2014-08-01

TM4SF5 overexpressed in hepatocellular carcinoma activates focal adhesion kinase (FAK) during tumor cell migration. However, it remains unknown how TM4SF5 in hepatocellular carcinoma cells compromises with immune actions initiated by extracellular cytokines. Normal and cancerous hepatocytes with or without TM4SF5 expression were analyzed for the effects of cytokine signaling activity on TM4SF5/FAK signaling and metastatic potential. We found that interleukin-6 (IL-6) was differentially expressed in hepatocytes depending on cancerous malignancy and TM4SF5 expression. IL-6 treatment activated FAK and STAT3 and enhanced focal adhesion (FA) formation in TM4SF5-null cells, but it decreased TM4SF5-dependent FAK activity and FA formation in SNU761-TM4SF5 cells. STAT3 suppression abolished the IL-6-mediated effects in normal Chang cells, but it did not recover the TM4SF5-dependent FAK activity that was inhibited by IL-6 treatment in cancerous SNU761-TM4SF5 cells. In addition, modulation of FAK activity did not change the IL-6-mediated STAT3 activity in either the Chang or SNU761 cell system. TM4SF5 expression in SNU761 cells caused invasive extracellular matrix degradation negatively depending on IL-6/IL-6 receptor (IL-6R) signaling. Thus, it is likely that hepatic cancer cells adopt TM4SF5-dependent FAK activation and metastatic potential by lowering IL-6 expression and avoiding its immunological action through the IL-6-STAT3 pathway. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Ubiquitination of the transcription factor IRF-3 activates RIPA, the apoptotic pathway that protects mice from viral pathogenesis

PubMed Central

Chattopadhyay, Saurabh; Kuzmanovic, Teodora; Zhang, Ying; Wetzel, Jaime L.; Sen, Ganes C.

2016-01-01

SUMMARY The transcription factor IRF-3 mediates cellular antiviral response by inducing the expression of interferon and other antiviral proteins. In RNA-virus infected cells, IRF-3’s transcriptional activation is triggered primarily by RIG-I-like receptors (RLR), which can also activate the RLR-induced IRF-3-mediated pathway of apoptosis (RIPA). Here, we have reported that the pathway of IRF-3 activation in RIPA was independent of and distinct from the known pathway of transcriptional activation of IRF-3. It required linear polyubiquitination of two specific lysine residues of IRF-3 by LUBAC, the linear polyubiquitinating enzyme complex, which bound IRF-3 in signal-dependent fashion. To evaluate the role of RIPA in viral pathogenesis, we engineered a genetically targeted mouse, which expressed a mutant IRF-3 that was RIPA-competent but transcriptionally inert; this single-action IRF-3 could protect mice from lethal viral infection. Our observations indicated that IRF-3-mediated apoptosis of virus-infected cells could be an effective antiviral mechanism, without expression of the interferon-stimulated genes. PMID:27178468

MicroRNA-214 controls skin and hair follicle development by modulating the activity of the Wnt pathway

PubMed Central

Ahmed, Mohammed I.; Alam, Majid; Emelianov, Vladimir U.; Poterlowicz, Krzysztof; Patel, Ankit; Sharov, Andrey A.; Mardaryev, Andrei N.

2014-01-01

Skin development is governed by complex programs of gene activation and silencing, including microRNA-dependent modulation of gene expression. Here, we show that miR-214 regulates skin morphogenesis and hair follicle (HF) cycling by targeting β-catenin, a key component of the Wnt signaling pathway. miR-214 exhibits differential expression patterns in the skin epithelium, and its inducible overexpression in keratinocytes inhibited proliferation, which resulted in formation of fewer HFs with decreased hair bulb size and thinner hair production. The inhibitory effects of miR-214 on HF development and cycling were associated with altered activities of multiple signaling pathways, including decreased expression of key Wnt signaling mediators β-catenin and Lef-1, and were rescued by treatment with pharmacological Wnt activators. Finally, we identify β-catenin as one of the conserved miR-214 targets in keratinocytes. These data provide an important foundation for further analyses of miR-214 as a key regulator of Wnt pathway activity and stem cell functions during normal tissue homeostasis, regeneration, and aging. PMID:25422376

Salmonella Biofilm Development Depends on the Phosphorylation Status of RcsB

PubMed Central

Latasa, Cristina; García, Begoña; Echeverz, Maite; Toledo-Arana, Alejandro; Valle, Jaione; Campoy, Susana; García-del Portillo, Francisco; Solano, Cristina

2012-01-01

The Rcs phosphorelay pathway is a complex signaling pathway involved in the regulation of many cell surface structures in enteric bacteria. In response to environmental stimuli, the sensor histidine kinase (RcsC) autophosphorylates and then transfers the phosphate through intermediary steps to the response regulator (RcsB), which, once phosphorylated, regulates gene expression. Here, we show that Salmonella biofilm development depends on the phosphorylation status of RcsB. Thus, unphosphorylated RcsB, hitherto assumed to be inactive, is essential to activate the expression of the biofilm matrix compounds. The prevention of RcsB phosphorylation either by the disruption of the phosphorelay at the RcsC or RcsD level or by the production of a nonphosphorylatable RcsB allele induces biofilm development. On the contrary, the phosphorylation of RcsB by the constitutive activation of the Rcs pathway inhibits biofilm development, an effect that can be counteracted by the introduction of a nonphosphorylatable RcsB allele. The inhibition of biofilm development by phosphorylated RcsB is due to the repression of CsgD expression, through a mechanism dependent on the accumulation of the small noncoding RNA RprA. Our results indicate that unphosphorylated RcsB plays an active role for integrating environmental signals and, more broadly, that RcsB phosphorylation acts as a key switch between planktonic and sessile life-styles in Salmonella enterica serovar Typhimurium. PMID:22582278

Anticancer mechanisms and clinical application of alkylphospholipids.

PubMed

van Blitterswijk, Wim J; Verheij, Marcel

2013-03-01

Synthetic alkylphospholipids (ALPs), such as edelfosine, miltefosine, perifosine, erucylphosphocholine and erufosine, represent a relatively new class of structurally related antitumor agents that act on cell membranes rather than on DNA. They selectively target proliferating (tumor) cells, inducing growth arrest and apoptosis, and are potent sensitizers of conventional chemo- and radiotherapy. ALPs easily insert in the outer leaflet of the plasma membrane and cross the membrane via an ATP-dependent CDC50a-containing 'flippase' complex (in carcinoma cells), or are internalized by lipid raft-dependent endocytosis (in lymphoma/leukemic cells). ALPs resist catabolic degradation, therefore accumulate in the cell and interfere with lipid-dependent survival signaling pathways, notably PI3K-Akt and Raf-Erk1/2, and de novo phospholipid biosynthesis. At the same time, stress pathways (e.g. stress-activated protein kinase/JNK) are activated to promote apoptosis. In many preclinical and clinical studies, perifosine was the most effective ALP, mainly because it inhibits Akt activity potently and consistently, also in vivo. This property is successfully exploited clinically in highly malignant tumors, such as multiple myeloma and neuroblastoma, in which a tyrosine kinase receptor/Akt pathway is amplified. In such cases, perifosine therapy is most effective in combination with conventional anticancer regimens or with rapamycin-type mTOR inhibitors, and may overcome resistance to these agents. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism. Copyright © 2012 Elsevier B.V. All rights reserved.

Prostaglandins inhibit 5-lipoxygenase-activating protein expression and leukotriene B4 production from dendritic cells via an IL-10-dependent mechanism.

PubMed

Harizi, Hedi; Juzan, Monique; Moreau, Jean-François; Gualde, Norbert

2003-01-01

PGs produced from arachidonic acid by the action of cyclooxygenase enzymes play a pivotal role in the regulation of both inflammatory and immune responses. Because leukotriene B4 (LTB4), a product of 5-lipoxygenase (5-LO) pathway, can exert numerous immunoregulatory and proinflammatory activities, we examined the effects of PGs on LTB4 release from dendritic cells (DC) and from peritoneal macrophages. In concentration-dependent manner, PGE1 and PGE2 inhibited the production of LTB4 from DC, but not from peritoneal macrophage, with an IC50 of 0.04 microM. The same effect was observed with MK-886, a 5-LO-activating protein (FLAP)-specific inhibitor. The decreased release of LTB4 was associated with an enhanced level of IL-10. Furthermore, the inhibition of LTB4 synthesis by PGs was significantly reversed by anti-IL-10, suggesting the involvement of an IL-10-dependent mechanism. Hence, we examined the effects of exogenous IL-10 on the 5-LO pathway. We demonstrate that IL-10 suppresses the production of LTB4 from DC by inhibiting FLAP protein expression without any effect on 5-LO and cytosolic phospholipase A2. Taken together, our results suggest links between DC cyclooxygenase and 5-LO pathways during the inflammatory response, and FLAP is a key target for the PG-induced IL-10-suppressive effects.

Ulex europaeus agglutinin II (UEA-II) is a novel, potent inhibitor of complement activation.

PubMed

Lekowski, R; Collard, C D; Reenstra, W R; Stahl, G L

2001-02-01

Complement is an important mediator of vascular injury following oxidative stress. We recently demonstrated that complement activation following endothelial oxidative stress is mediated by mannose-binding lectin (MBL) and activation of the lectin complement pathway. Here, we investigated whether nine plant lectins which have a binding profile similar to that of MBL competitively inhibit MBL deposition and subsequent complement activation following human umbilical vein endothelial cell (HUVEC) oxidative stress. HUVEC oxidative stress (1% O(2), 24 hr) significantly increased Ulex europaeus agglutinin II (UEA-II) binding by 72 +/- 9% compared to normoxic cells. UEA-II inhibited MBL binding to HUVEC in a concentration-dependent manner following oxidative stress. Further, MBL inhibited UEA-II binding to HUVEC in a concentration-dependent manner following oxidative stress, suggesting a common ligand. UEA-II (< or = 100 micromol/L) did not attenuate the hemolytic activity, nor did it inhibit C3a des Arg formation from alternative or classical complement pathway-specific hemolytic assays. C3 deposition (measured by ELISA) following HUVEC oxidative stress was inhibited by UEA-II in a concentration-dependent manner (IC(50) = 10 pmol/L). UEA-II inhibited C3 and MBL co-localization (confocal microscopy) in a concentration-dependent manner on HUVEC following oxidative stress (IC(50) approximately 1 pmol/L). Finally, UEA-II significantly inhibited complement-dependent neutrophil chemotaxis, but failed to inhibit fMLP-mediated chemotaxis, following endothelial oxidative stress. These data demonstrate that UEA-II is a novel, potent inhibitor of human MBL deposition and complement activation following human endothelial oxidative stress.

Ulex europaeus agglutinin II (UEA-II) is a novel, potent inhibitor of complement activation

PubMed Central

Lekowski, Robert; Collard, Charles D.; Reenstra, Wende R.; Stahl, Gregory L.

2001-01-01

Complement is an important mediator of vascular injury following oxidative stress. We recently demonstrated that complement activation following endothelial oxidative stress is mediated by mannose-binding lectin (MBL) and activation of the lectin complement pathway. Here, we investigated whether nine plant lectins which have a binding profile similar to that of MBL competitively inhibit MBL deposition and subsequent complement activation following human umbilical vein endothelial cell (HUVEC) oxidative stress. HUVEC oxidative stress (1% O2, 24 hr) significantly increased Ulex europaeus agglutinin II (UEA-II) binding by 72 ± 9% compared to normoxic cells. UEA-II inhibited MBL binding to HUVEC in a concentration-dependent manner following oxidative stress. Further, MBL inhibited UEA-II binding to HUVEC in a concentration-dependent manner following oxidative stress, suggesting a common ligand. UEA-II (≤ 100 μmol/L) did not attenuate the hemolytic activity, nor did it inhibit C3a des Arg formation from alternative or classical complement pathway-specific hemolytic assays. C3 deposition (measured by ELISA) following HUVEC oxidative stress was inhibited by UEA-II in a concentration-dependent manner (IC50 = 10 pmol/L). UEA-II inhibited C3 and MBL co-localization (confocal microscopy) in a concentration-dependent manner on HUVEC following oxidative stress (IC50 ≈ 1 pmol/L). Finally, UEA-II significantly inhibited complement-dependent neutrophil chemotaxis, but failed to inhibit fMLP-mediated chemotaxis, following endothelial oxidative stress. These data demonstrate that UEA-II is a novel, potent inhibitor of human MBL deposition and complement activation following human endothelial oxidative stress. PMID:11266613

Involvement of TrkB- and p75NTR-signaling pathways in two contrasting forms of long-lasting synaptic plasticity

NASA Astrophysics Data System (ADS)

Sakuragi, Shigeo; Tominaga-Yoshino, Keiko; Ogura, Akihiko

2013-11-01

The repetition of experience is often necessary to establish long-lasting memory. However, the cellular mechanisms underlying this repetition-dependent consolidation of memory remain unclear. We previously observed in organotypic slice cultures of the rodent hippocampus that repeated inductions of long-term potentiation (LTP) led to a slowly developing long-lasting synaptic enhancement coupled with synaptogenesis. We also reported that repeated inductions of long-term depression (LTD) produced a long-lasting synaptic suppression coupled with synapse elimination. We proposed these phenomena as useful in vitro models for analyzing repetition-dependent consolidation. Here, we hypothesized that the enhancement and suppression are mediated by the brain-derived neurotrophic factor (BDNF)-TrkB signaling pathway and the proBDNF-p75NTR pathway, respectively. When we masked the respective pathways, reversals of the enhancement and suppression resulted. These results suggest the alternative activation of the p75NTR pathway by BDNF under TrkB-masking conditions and of the TrkB pathway by proBDNF under p75NTR-masking conditions, thus supporting the aforementioned hypothesis.

Sex-specific activation of cell death signalling pathways in cerebellar granule neurons exposed to oxygen glucose deprivation followed by reoxygenation

PubMed Central

Sharma, Jaswinder; Nelluru, Geetha; Ann Wilson, Mary; Johnston, Michael V; Ahamed Hossain, Mir

2011-01-01

Neuronal death pathways following hypoxia–ischaemia are sexually dimorphic, but the underlying mechanisms are unclear. We examined cell death mechanisms during OGD (oxygen-glucose deprivation) followed by Reox (reoxygenation) in segregated male (XY) and female (XX) mouse primary CGNs (cerebellar granule neurons) that are WT (wild-type) or Parp-1 [poly(ADP-ribose) polymerase 1] KO (knockout). Exposure of CGNs to OGD (1.5 h)/Reox (7 h) caused cell death in XY and XX neurons, but cell death during Reox was greater in XX neurons. ATP levels were significantly lower after OGD/Reox in WT-XX neurons than in XY neurons; this difference was eliminated in Parp-1 KO-XX neurons. AIF (apoptosis-inducing factor) was released from mitochondria and translocated to the nucleus by 1 h exclusively in WT-XY neurons. In contrast, there was a release of Cyt C (cytochrome C) from mitochondria in WT-XX and Parp-1 KO neurons of both sexes; delayed activation of caspase 3 was observed in the same three groups. Thus deletion of Parp-1 shunted cell death towards caspase 3-dependent apoptosis. Delayed activation of caspase 8 was also observed in all groups after OGD/Reox, but was much greater in XX neurons, and caspase 8 translocated to the nucleus in XX neurons only. Caspase 8 activation may contribute to increased XX neuronal death during Reox, via caspase 3 activation. Thus, OGD/Reox induces death of XY neurons via a PARP-1-AIF-dependent mechanism, but blockade of PARP-1-AIF pathway shifts neuronal death towards a caspase-dependent mechanism. In XX neurons, OGD/Reox caused prolonged depletion of ATP and delayed activation of caspase 8 and caspase 3, culminating in greater cell death during Reox. PMID:21382016

Modulation of Estrogen Response Element-Driven Gene Expressions and Cellular Proliferation with Polar Directions by Designer Transcription Regulators

PubMed Central

Muyan, Mesut; Güpür, Gizem; Yaşar, Pelin; Ayaz, Gamze; User, Sırma Damla; Kazan, Hasan Hüseyin; Huang, Yanfang

2015-01-01

Estrogen receptor α (ERα), as a ligand-dependent transcription factor, mediates 17β-estradiol (E2) effects. ERα is a modular protein containing a DNA binding domain (DBD) and transcription activation domains (AD) located at the amino- and carboxyl-termini. The interaction of the E2-activated ERα dimer with estrogen response elements (EREs) of genes constitutes the initial step in the ERE-dependent signaling pathway necessary for alterations of cellular features. We previously constructed monomeric transcription activators, or monotransactivators, assembled from an engineered ERE-binding module (EBM) using the ERα-DBD and constitutively active ADs from other transcription factors. Monotransactivators modulated cell proliferation by activating and repressing ERE-driven gene expressions that simulate responses observed with E2-ERα. We reasoned here that integration of potent heterologous repression domains (RDs) into EBM could generate monotransrepressors that alter ERE-bearing gene expressions and cellular proliferation in directions opposite to those observed with E2-ERα or monotransactivators. Consistent with this, monotransrepressors suppressed reporter gene expressions that emulate the ERE-dependent signaling pathway. Moreover, a model monotransrepressor regulated DNA synthesis, cell cycle progression and proliferation of recombinant adenovirus infected ER-negative cells through decreasing as well as increasing gene expressions with polar directions compared with E2-ERα or monotransactivator. Our results indicate that an ‘activator’ or a ‘repressor’ possesses both transcription activating/enhancing and repressing/decreasing abilities within a chromatin context. Offering a protein engineering platform to alter signal pathway-specific gene expressions and cell growth, our approach could also be used for the development of tools for epigenetic modifications and for clinical interventions wherein multigenic de-regulations are an issue. PMID:26295471

Membrane glucocorticoid receptors are localised in the extracellular matrix and signal through the MAPK pathway in mammalian skeletal muscle fibres

PubMed Central

Boncompagni, Simona; Arthurton, Lewis; Akujuru, Eugene; Pearson, Timothy; Steverding, Dietmar; Protasi, Feliciano; Mutungi, Gabriel

2015-01-01

A number of studies have previously proposed the existence of glucocorticoid receptors on the plasma membrane of many cell types, including skeletal muscle fibres. However, their exact localisation and the cellular signalling pathway(s) they utilise to communicate with the rest of the cell are still poorly understood. In this study, we investigated the localisation and the mechanism(s) underlying the non-genomic physiological functions of these receptors in mouse skeletal muscle cells. The results show that the receptors were localised in the cytoplasm in myoblasts, in the nucleus in myotubes, in the extracellular matrix, in satellite cells and in the proximity of mitochondria in adult muscle fibres. Also, they bound laminin in a glucocorticoid-dependent manner. Treating small skeletal muscle fibre bundles with the synthetic glucocorticoid beclomethasone dipropionate increased the phosphorylation (= activation) of extracellular signal-regulated kinases 1 and 2, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase. This occurred within 5 min and depended on the fibre type and the duration of the treatment. It was also abolished by the glucocorticoid receptor inhibitor, mifepristone, and a monoclonal antibody against the receptor. From these results we conclude that the non-genomic/non-canonical physiological functions of glucocorticoids, in adult skeletal muscle fibres, are mediated by a glucocorticoid receptor localised in the extracellular matrix, in satellite cells and close to mitochondria, and involve activation of the mitogen-activated protein kinase pathway. PMID:25846902

Tl(I) and Tl(III) activate both mitochondrial and extrinsic pathways of apoptosis in rat pheochromocytoma (PC12) cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hanzel, Cecilia Eliana; Verstraeten, Sandra Viviana

2009-04-01

Thallium (Tl) is a highly toxic metal though yet its mechanisms are poorly understood. Previously, we demonstrated that rat pheochromocytoma (PC12) cells exposure to thallous (Tl(I)) or thallic (Tl(III)) cations leads to mitochondrial damage and reduced cell viability. In the present work we comparatively characterized the possible pathways involved in Tl(I)- and Tl(III)- (10-100 {mu}M) mediated decrease in PC12 cells viability. We observed that these cations do not cause cell necrosis but significantly increased the number of cells with apoptotic features. Both cations lead to Bax oligomerization and caused apoptosis inducing factor (AIF), endonuclease G (Endo G), and cytochrome cmore » release from mitochondria, but they did not activate caspase dependent DNAse (CAD). Tl(I)- and Tl(III)-dependent caspases 9 and 3 activation followed similar kinetics, with maximal effects at 18 h of incubation. In addition, Tl(I) promoted phosphatidylserine (PS) exposure. Tl(III) induced 2- and 18-fold increase in Fas content and caspase 8 activity, respectively. Together, experimental results show that Tl(I) and Tl(III) induce PC12 cells apoptosis, although differential pathways are involved. While Tl(I)-mediated cell apoptosis was mainly associated with mitochondrial damage, Tl(III) showed a mixed effect triggering both the intrinsic and extrinsic pathways of apoptosis. These findings contribute to a better understanding of the mechanisms underlying Tl-induced loss of cell viability in PC12 cells.« less

Distinct Signaling Pathways Mediate Stimulation of Cell Cycle Progression and Prevention of Apoptotic Cell Death by Estrogen in Rat Pituitary Tumor PR1 Cells

PubMed Central

Caporali, Simona; Imai, Manami; Altucci, Lucia; Cancemi, Massimo; Caristi, Silvana; Cicatiello, Luigi; Matarese, Filomena; Penta, Roberta; Sarkar, Dipak K.; Bresciani, Francesco; Weisz, Alessandro

2003-01-01

Estrogens control cell growth and viability in target cells via an interplay of genomic and extragenomic pathways not yet elucidated. Here, we show evidence that cell proliferation and survival are differentially regulated by estrogen in rat pituitary tumor PR1 cells. Pico- to femtomolar concentrations of 17β-estradiol (E2) are sufficient to foster PR1 cell proliferation, whereas nanomolar concentrations of the same are needed to prevent cell death that occurs at a high rate in these cells in the absence of hormone. Activation of endogenous (PRL) or transfected estrogen-responsive genes occurs at the same, higher concentrations of E2 required to promote cell survival, whereas stimulation of cyclin D3 expression and DNA synthesis occur at lower E2 concentrations. Similarly, the pure antiestrogen ICI 182,780 inhibits estrogen response element-dependent trans-activation and cell death more effectively than cyclin-cdk activity, G1-S transition, or DNA synthesis rate. In antiestrogen-treated and/or estrogen-deprived cells, death is due predominantly to apoptosis. Estrogen-induced cell survival, but not E2-dependent cell cycle progression, can be prevented by an inhibitor of c-Src kinase or by blockade of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signaling pathway. These data indicate the coexistence of two distinguishable estrogen signaling pathways in PR1 cells, characterized by different functions and sensitivity to hormones and antihormones. PMID:12960425

Caffeine Inhibits the Activation of Hepatic Stellate Cells Induced by Acetaldehyde via Adenosine A2A Receptor Mediated by the cAMP/PKA/SRC/ERK1/2/P38 MAPK Signal Pathway

PubMed Central

Yang, Wanzhi; Wang, Qi; Zhao, Han; Yang, Feng; Lv, Xiongwen; Li, Jun

2014-01-01

Hepatic stellate cell (HSC) activation is an essential event during alcoholic liver fibrosis. Evidence suggests that adenosine aggravates liver fibrosis via the adenosine A2A receptor (A2AR). Caffeine, which is being widely consumed during daily life, inhibits the action of adenosine. In this study, we attempted to validate the hypothesis that caffeine influences acetaldehyde-induced HSC activation by acting on A2AR. Acetaldehyde at 50, 100, 200, and 400 μM significantly increased HSC-T6 cells proliferation, and cell proliferation reached a maximum at 48 h after exposure to 200 μM acetaldehyde. Caffeine and the A2AR antagonist ZM241385 decreased the cell viability and inhibited the expression of procollagen type I and type III in acetaldehyde-induced HSC-T6 cells. In addition, the inhibitory effect of caffeine on the expression of procollagen type I was regulated by A2AR-mediated signal pathway involving cAMP, PKA, SRC, and ERK1/2. Interestingly, caffeine’s inhibitory effect on the expression of procollagen type III may depend upon the A2AR-mediated P38 MAPK-dependent pathway. Conclusions: Caffeine significantly inhibited acetaldehyde-induced HSC-T6 cells activation by distinct A2AR mediated signal pathway via inhibition of cAMP-PKA-SRC-ERK1/2 for procollagen type I and via P38 MAPK for procollagen type III. PMID:24682220

Activation of PTHrP-cAMP-CREB1 signaling following p53 loss is essential for osteosarcoma initiation and maintenance

PubMed Central

Walia, Mannu K; Ho, Patricia MW; Taylor, Scott; Ng, Alvin JM; Gupte, Ankita; Chalk, Alistair M; Zannettino, Andrew CW; Martin, T John; Walkley, Carl R

2016-01-01

Mutations in the P53 pathway are a hallmark of human cancer. The identification of pathways upon which p53-deficient cells depend could reveal therapeutic targets that may spare normal cells with intact p53. In contrast to P53 point mutations in other cancer, complete loss of P53 is a frequent event in osteosarcoma (OS), the most common cancer of bone. The consequences of p53 loss for osteoblastic cells and OS development are poorly understood. Here we use murine OS models to demonstrate that elevated Pthlh (Pthrp), cAMP levels and signalling via CREB1 are characteristic of both p53-deficient osteoblasts and OS. Normal osteoblasts survive depletion of both PTHrP and CREB1. In contrast, p53-deficient osteoblasts and OS depend upon continuous activation of this pathway and undergo proliferation arrest and apoptosis in the absence of PTHrP or CREB1. Our results identify the PTHrP-cAMP-CREB1 axis as an attractive pathway for therapeutic inhibition in OS. DOI: http://dx.doi.org/10.7554/eLife.13446.001 PMID:27070462

Glucose dependence of glycogen synthase activity regulation by GSK3 and MEK/ERK inhibitors and angiotensin-(1-7) action on these pathways in cultured human myotubes.

PubMed

Montori-Grau, Marta; Tarrats, Núria; Osorio-Conles, Oscar; Orozco, Anna; Serrano-Marco, Lucía; Vázquez-Carrera, Manuel; Gómez-Foix, Anna M

2013-05-01

Glycogen synthase (GS) is activated by glucose/glycogen depletion in skeletal muscle cells, but the contributing signaling pathways, including the chief GS regulator GSK3, have not been fully defined. The MEK/ERK pathway is known to regulate GSK3 and respond to glucose. The aim of this study was to elucidate the GSK3 and MEK/ERK pathway contribution to GS activation by glucose deprivation in cultured human myotubes. Moreover, we tested the glucose-dependence of GSK3 and MEK/ERK effects on GS and angiotensin (1-7) actions on these pathways. We show that glucose deprivation activated GS, but did not change phospho-GS (Ser640/1), GSK3β activity or activity-activating phosphorylation of ERK1/2. We then treated glucose-replete and -depleted cells with SB415286, U0126, LY294 and rapamycin to inhibit GSK3, MEK1/2, PI3K and mTOR, respectively. SB415286 activated GS and decreased the relative phospho-GS (Ser640/1) level, more in glucose-depleted than -replete cells. U0126 activated GS and reduced the phospho-GS (Ser640/1) content significantly in glucose-depleted cells, while GSK3β activity tended to increase. LY294 inactivated GS in glucose-depleted cells only, without affecting relative phospho-GS (Ser640/1) level. Rapamycin had no effect on GS activation. Angiotensin-(1-7) raised phospho-ERK1/2 but not phospho-GSK3β (Ser9) content, while it inactivated GS and increased GS phosphorylation on Ser640/1, in glucose-replete cells. In glucose-depleted cells, angiotensin-(1-7) effects on ERK1/2 and GS were reverted, while relative phospho-GSK3β (Ser9) content decreased. In conclusion, activation of GS by glucose deprivation is not due to GS Ser640/1 dephosphorylation, GSK3β or ERK1/2 regulation in cultured myotubes. However, glucose depletion enhances GS activation/Ser640/1 dephosphorylation due to both GSK3 and MEK/ERK inhibition. Angiotensin-(1-7) inactivates GS in glucose-replete cells in association with ERK1/2 activation, not with GSK3 regulation, and glucose deprivation reverts both hormone effects. Thus, the ERK1/2 pathway negatively regulates GS activity in myotubes, without involving GSK3 regulation, and as a function of the presence of glucose. Copyright © 2013 Elsevier Inc. All rights reserved.

Silencing of Pokemon enhances caspase-dependent apoptosis via fas- and mitochondria-mediated pathways in hepatocellular carcinoma cells.

PubMed

Zhang, Yu-Qin; Xiao, Chuan-Xing; Lin, Bi-Yun; Shi, Ying; Liu, Yun-Peng; Liu, Jing-Jing; Guleng, Bayasi; Ren, Jian-Lin

2013-01-01

The role of Pokemon (POK erythroid myeloid ontogenic actor), a recently identified POK transcription factor with proto-oncogenic activity, in hepatocellular carcinogenesis has only been assessed by a few studies. Our previous study revealed that Pokemon is overexpressed in hepatocellular carcinomas (HCC) and promotes HCC cell proliferation and migration via an AKT- and ERK- dependent manner. In the present study, we used the TUNEL assay and FACS analysis to demonstrate that oxaliplatin induced apoptosis was significantly increased in cells with silenced Pokemon. Western blots showed that p53 expression and phosphorylation were significantly increased in Pokemon defective cells, thereby initiating the mitochondria-mediated and death receptor-mediated apoptotic pathways. In the mitochondria-mediated pathway, expression of pro-apoptotic Bcl-2 family members (including Bad, Bid, Bim and Puma) as well as AIF was increased and decreasing the mitochondrial membrane potential resulted in cytochrome C released from mitochondrial in HepG2 si-Pokemon cells. In addition, upon oxaliplatin treatment of Pokemon-silenced cells, the FAS receptor, FADD and their downstream targets caspase-10 and caspase-8 were activated, causing increased release of caspase-8 active fragments p18 and p10. Increased activated caspase-8-mediated cleavage and activation of downstream effector caspases such as caspase-9 and caspase-3 was observed in HepG2 si-Pokemon cells as compared to control. Therefore, Pokemon might serve as an important mediator of crosstalk between intrinsic and extrinsic apoptotic pathways in HCC cells. Moreover, our findings suggest that Pokemon could be an attractive therapeutic target gene for human cancer therapy.

Silencing of Pokemon Enhances Caspase-Dependent Apoptosis via Fas- and Mitochondria-Mediated Pathways in Hepatocellular Carcinoma Cells

PubMed Central

Lin, Bi-Yun; Shi, Ying; Liu, Yun-Peng; Liu, Jing-Jing; Guleng, Bayasi; Ren, Jian-Lin

2013-01-01

The role of Pokemon (POK erythroid myeloid ontogenic actor), a recently identified POK transcription factor with proto-oncogenic activity, in hepatocellular carcinogenesis has only been assessed by a few studies. Our previous study revealed that Pokemon is overexpressed in hepatocellular carcinomas (HCC) and promotes HCC cell proliferation and migration via an AKT- and ERK- dependent manner. In the present study, we used the TUNEL assay and FACS analysis to demonstrate that oxaliplatin induced apoptosis was significantly increased in cells with silenced Pokemon. Western blots showed that p53 expression and phosphorylation were significantly increased in Pokemon defective cells, thereby initiating the mitochondria-mediated and death receptor-mediated apoptotic pathways. In the mitochondria-mediated pathway, expression of pro-apoptotic Bcl-2 family members (including Bad, Bid, Bim and Puma) as well as AIF was increased and decreasing the mitochondrial membrane potential resulted in cytochrome C released from mitochondrial in HepG2 si-Pokemon cells. In addition, upon oxaliplatin treatment of Pokemon-silenced cells, the FAS receptor, FADD and their downstream targets caspase-10 and caspase-8 were activated, causing increased release of caspase-8 active fragments p18 and p10. Increased activated caspase-8-mediated cleavage and activation of downstream effector caspases such as caspase-9 and caspase-3 was observed in HepG2 si-Pokemon cells as compared to control. Therefore, Pokemon might serve as an important mediator of crosstalk between intrinsic and extrinsic apoptotic pathways in HCC cells. Moreover, our findings suggest that Pokemon could be an attractive therapeutic target gene for human cancer therapy. PMID:23874836

The progesterone-induced enhancement of object recognition memory consolidation involves activation of the extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin (mTOR) pathways in the dorsal hippocampus

PubMed Central

Orr, Patrick T.; Rubin, Amanda J.; Fan, Lu; Kent, Brianne A.; Frick, Karyn M.

2012-01-01

Although much recent work has elucidated the biochemical mechanisms underlying the modulation of memory by 17β-estradiol, little is known about the signaling events through which progesterone (P) regulates memory. We recently demonstrated that immediate post-training infusion of P into the dorsal hippocampus enhances object recognition memory consolidation in young ovariectomized female mice (Orr et al., 2009). The goal of the present study was to identify the biochemical alterations that might underlie this mnemonic enhancement. We hypothesized that the P-induced enhancement of object recognition would be dependent on activation of the ERK and mTOR pathways. In young ovariectomized mice, we found that bilateral dorsal hippocampal infusion of P significantly increased levels of phospho-p42 ERK and the mTOR substrate S6K in the dorsal hippocampus 5 minutes after infusion. Phospho-p42 ERK levels were downregulated 15 minutes after infusion and returned to baseline 30 minutes after infusion, suggesting a biphasic effect of P on ERK activation. Dorsal hippocampal ERK and mTOR activation were necessary for P to facilitate memory consolidation, as suggested by the fact that inhibitors of both pathways infused into the dorsal hippocampus immediately after training blocked the P-induced enhancement of object recognition. Collectively, these data provide the first demonstration that the ability of P to enhance memory consolidation depends on the rapid activation of cell signaling and protein synthesis pathways in the dorsal hippocampus. PMID:22265866

TAK1 regulates NF-{Kappa}B and AP-1 activation in airway epithelial cells following RSV infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dey, Nilay; Liu Tianshuang; Garofalo, Roberto P.

2011-09-30

Respiratory syncytial virus (RSV) is the most common cause of epidemic respiratory diseases in infants and young children. RSV infection of airway epithelial cells induces the expression of immune/inflammatory genes through the activation of a subset of transcription factors, including Nuclear Factor-{kappa}B (NF-{kappa}B) and AP-1. In this study, we have investigated the signaling pathway leading to activation of these two transcription factors in response to RSV infection. Our results show that IKK{beta} plays a key role in viral-induced NF-{kappa}B activation, while JNK regulates AP-1-dependent gene transcription, as demonstrated by using kinase inactive proteins and chemical inhibitors of the two kinases.more » Inhibition of TAK1 activation, by overexpression of kinase inactive TAK1 or using cells lacking TAK1 expression, significantly reduced RSV-induced NF-{kappa}B and AP-1 nuclear translocation and DNA-binding activity, as well as NF-{kappa}B-dependent gene expression, identifying TAK1 as an important upstream signaling molecule regulating RSV-induced NF-{kappa}B and AP-1 activation. - Highlights: > IKK{beta} is a major kinase involved in RSV-induced NF-{kappa}B activation. > JNK regulates AP-1-dependent gene transcription in RSV infection. > TAK1 is a critical upstream signaling molecule for both pathways in infected cells.« less

AM fungal exudates activate MAP kinases in plant cells in dependence from cytosolic Ca(2+) increase.

PubMed

Francia, Doriana; Chiltz, Annick; Lo Schiavo, Fiorella; Pugin, Alain; Bonfante, Paola; Cardinale, Francesca

2011-09-01

The molecular dialogue occurring prior to direct contact between the fungal and plant partners of arbuscular-mycorrhizal (AM) symbioses begins with the release of fungal elicitors, so far only partially identified chemically, which can activate specific signaling pathways in the host plant. We show here that the activation of MAPK is also induced by exudates of germinating spores of Gigaspora margarita in cultured cells of the non-leguminous species tobacco (Nicotiana tabacum), as well as in those of the model legume Lotus japonicus. MAPK activity peaked about 15 min after the exposure of the host cells to the fungal exudates (FE). FE were also responsible for a rapid and transient increase in free cytosolic Ca(2+) in Nicotiana plumbaginifolia and tobacco cells, and pre-treatment with a Ca(2+)-channel blocker (La(3+)) showed that in these cells, MAPK activation was dependent on the cytosolic Ca(2+) increase. A partial dependence of MAPK activity on the common Sym pathway could be demonstrated for a cell line of L. japonicus defective for LjSym4 and hence unable to establish an AM symbiosis. Our results show that MAPK activation is triggered by an FE-induced cytosolic Ca(2+) transient, and that a Sym genetic determinant acts to modulate the intensity and duration of this activity. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Signal-transducing protein phosphorylation cascades mediated by Ras/Rho proteins in the mammalian cell: the potential for multiplex signalling.

PubMed Central

Denhardt, D T

1996-01-01

The features of three distinct protein phosphorylation cascades in mammalian cells are becoming clear. These signalling pathways link receptor-mediated events at the cell surface or intracellular perturbations such as DNA damage to changes in cytoskeletal structure, vesicle transport and altered transcription factor activity. The best known pathway, the Ras-->Raf-->MEK-->ERK cascade [where ERK is extracellular-signal-regulated kinase and MEK is mitogen-activated protein (MAP) kinase/ERK kinase], is typically stimulated strongly by mitogens and growth factors. The other two pathways, stimulated primarily by assorted cytokines, hormones and various forms of stress, predominantly utilize p21 proteins of the Rho family (Rho, Rac and CDC42), although Ras can also participate. Diagnostic of each pathway is the MAP kinase component, which is phosphorylated by a unique dual-specificity kinase on both tyrosine and threonine in one of three motifs (Thr-Glu-Tyr, Thr-Phe-Tyr or Thr-Gly-Tyr), depending upon the pathway. In addition to activating one or more protein phosphorylation cascades, the initiating stimulus may also mobilize a variety of other signalling molecules (e.g. protein kinase C isoforms, phospholipid kinases, G-protein alpha and beta gamma subunits, phospholipases, intracellular Ca2+). These various signals impact to a greater or lesser extent on multiple downstream effectors. Important concepts are that signal transmission often entails the targeted relocation of specific proteins in the cell, and the reversible formation of protein complexes by means of regulated protein phosphorylation. The signalling circuits may be completed by the phosphorylation of upstream effectors by downstream kinases, resulting in a modulation of the signal. Signalling is terminated and the components returned to the ground state largely by dephosphorylation. There is an indeterminant amount of cross-talk among the pathways, and many of the proteins in the pathways belong to families of closely related proteins. The potential for more than one signal to be conveyed down a pathway simultaneously (multiplex signalling) is discussed. The net effect of a given stimulus on the cell is the result of a complex intracellular integration of the intensity and duration of activation of the individual pathways. The specific outcome depends on the particular signalling molecules expressed by the target cells and on the dynamic balance among the pathways. PMID:8836113

Glutamate-dependent phosphorylation of the mammalian target of rapamycin (mTOR) in Bergmann glial cells.

PubMed

Zepeda, Rossana C; Barrera, Iliana; Castelán, Francisco; Suárez-Pozos, Edna; Melgarejo, Yaaziel; González-Mejia, Elba; Hernández-Kelly, Luisa C; López-Bayghen, Esther; Aguilera, José; Ortega, Arturo

2009-09-01

Glutamate, the major excitatory neurotransmitter in the mammalian central nervous system, plays an important role in neuronal development and synaptic plasticity. It activates a variety of signaling pathways that regulate gene expression at the transcriptional and translational levels. Within glial cells, besides transcription, glutamate also regulates translation initiation and elongation. The mammalian target of rapamycin (mTOR), a key participant in the translation process, represents an important regulatory locus for translational control. Therefore, in the present communication we sought to characterize the mTOR phosphorylation pattern after glutamate treatment in chick cerebellar Bergmann glia primary cultures. A time- and dose-dependent increase in mTOR Ser 2448 phosphorylation was found. Pharmacological tools established that the glutamate effect is mediated through ionotropic and metabotropic receptors and interestingly, the glutamate transporter system is also involved. The signaling cascade triggered by glutamate includes an increase in intracellular Ca2+ levels, and the activation of the p60(Src)/PI-3K/PKB pathway. These results suggest that glia cells participate in the activity-dependent change in the brain protein repertoire.

Sarsaparilla (Smilax Glabra Rhizome) Extract Activates Redox-Dependent ATM/ATR Pathway to Inhibit Cancer Cell Growth by S Phase Arrest, Apoptosis, and Autophagy.

PubMed

She, Tiantian; Feng, Junnan; Lian, Shenyi; Li, Ruobing; Zhao, Chuanke; Song, Guoliang; Luo, Jie; Dawuti, Rouxianguli; Cai, Shaoqing; Qu, Like; Shou, Chengchao

2017-01-01

Sarsaparilla (Smilax Glabra Rhizome) exerts growth inhibitory effect on multiple cancer cells in vitro and in vivo, and redox-dependent persistent activation of ERK1/2 has been reported to underlie this effect. Here, we report an activation of ATM/ATR-dependent signaling pathway also as a mechanism for the cancer cell growth inhibition induced by the supernatant fraction of the water-soluble extract from sarsaparilla (SW). SW treatment (3.5 μg/μL) promoted the phosphorylations of ATM, ATR, and CHK1 in AGS and HT-29 cells. The ATM kinase inhibitor, KU55933, could reverse SW-induced ERK phosphorylation but not the reduced glutathione/oxidized glutathione (GSH/GSSG) imbalance in AGS cells. However, both the redox inhibitor glutathione (GSH) and ERK inhibitor U0126 antagonized SW-induced phosphorylations of ATM, ATR, and CHK1 in AGS cells. We further found KU55933 significantly antagonized SW-induced S phase arrest, apoptosis, autophagy and the resultant cell growth inhibition. Our results provide another molecular basis for the anticancer action of sarsaparilla.

Ribosomal stress induces L11- and p53-dependent apoptosis in mouse pluripotent stem cells.

PubMed

Morgado-Palacin, Lucia; Llanos, Susana; Serrano, Manuel

2012-02-01

Ribosome biogenesis is the most demanding energetic process in proliferating cells and it is emerging as a critical sensor of cellular homeostasis. Upon disturbance of ribosome biogenesis, specific free ribosomal proteins, most notably L11, bind and inhibit Mdm2, resulting in activation of the tumor suppressor p53. This pathway has been characterized in somatic and cancer cells, but its function in embryonic pluripotent cells has remained unexplored. Here, we show that treatment with low doses of Actinomycin D or depletion of ribosomal protein L37, two well-established inducers of ribosomal stress, activate p53 in an L11-dependent manner in mouse embryonic stem cells (ESCs) and in induced pluripotent stem cells (iPSCs). Activation of p53 results in transcriptional induction of p53 targets, including p21, Mdm2, Pidd, Puma, Noxa and Bax. Finally, ribosomal stress elicits L11- and p53-dependent apoptosis in ESCs/iPSCs. These results extend to pluripotent cells the functionality of the ribosomal stress pathway and we speculate that this could be a relevant cellular checkpoint during early embryogenesis.

Massive palmitoylation-dependent endocytosis during reoxygenation of anoxic cardiac muscle

PubMed Central

Lin, Mei-Jung; Fine, Michael; Lu, Jui-Yun; Hofmann, Sandra L; Frazier, Gary; Hilgemann, Donald W

2013-01-01

In fibroblasts, large Ca transients activate massive endocytosis (MEND) that involves membrane protein palmitoylation subsequent to mitochondrial permeability transition pore (PTP) openings. Here, we characterize this pathway in cardiac muscle. Myocytes with increased expression of the acyl transferase, DHHC5, have decreased Na/K pump activity. In DHHC5-deficient myocytes, Na/K pump activity and surface area/volume ratios are increased, the palmitoylated regulatory protein, phospholemman (PLM), and the cardiac Na/Ca exchanger (NCX1) show greater surface membrane localization, and MEND is inhibited in four protocols. Both electrical and optical methods demonstrate that PTP-dependent MEND occurs during reoxygenation of anoxic hearts. Post-anoxia MEND is ablated in DHHC5-deficient hearts, inhibited by cyclosporine A (CsA) and adenosine, promoted by staurosporine (STS), reduced in hearts lacking PLM, and correlates with impaired post-anoxia contractile function. Thus, the MEND pathway appears to be deleterious in severe oxidative stress but may constitutively contribute to cardiac sarcolemma turnover in dependence on metabolic stress. DOI: http://dx.doi.org/10.7554/eLife.01295.001 PMID:24282237

Activation of mitogen-activated protein kinase/extracellular signal-regulated kinase in hippocampal circuitry is required for consolidation and reconsolidation of recognition memory.

PubMed

Kelly, Aine; Laroche, Serge; Davis, Sabrina

2003-06-15

Consolidation and reconsolidation of long-term memory have been shown to be dependent on the synthesis of new proteins, but the specific molecular mechanisms underlying these events remain to be elucidated. The mitogen-activated protein kinase (MAPK) pathway can trigger genomic responses in neurons, leading to changes in protein synthesis, and several studies have identified its pivotal role in synaptic plasticity and long-term memory formation. In this study, we analyze the involvement of this pathway in the consolidation and reconsolidation of long-term recognition memory, using an object recognition task. We show that inhibition of the MAPK pathway by intracerebroventricular injection of the MEK [MAPK/extracellular signal-regulated kinase (ERK)] inhibitor UO126 blocks consolidation of object recognition memory but does not affect short-term memory. Brain regions of the entorhinal cortex-hippocampal circuitry were analyzed for ERK activation, and it was shown that consolidation of recognition memory was associated with increased phosphorylation of ERK in the dentate gyrus and entorhinal cortex, although total expression of ERK was unchanged. We also report that inhibition of the MAPK pathway blocks reconsolidation of recognition memory, and this was shown to be dependent on reactivation of the memory trace by brief reexposure to the objects. In addition, reconsolidation of memory was associated with an increase in the phosphorylation of ERK in entorhinal cortex and CA1. In summary, our data show that the MAPK kinase pathway is required for both consolidation and reconsolidation of long-term recognition memory, and that this is associated with hyperphosphorylation of ERK in different subregions of the entorhinal cortex-hippocampal circuitry.

Mannose Binding Lectin Is Required for Alphavirus-Induced Arthritis/Myositis

PubMed Central

Whitmore, Alan C.; Blevins, Lance K.; Hueston, Linda; Fraser, Robert J.; Herrero, Lara J.; Ramirez, Ruben; Smith, Paul N.; Mahalingam, Suresh; Heise, Mark T.

2012-01-01

Mosquito-borne alphaviruses such as chikungunya virus and Ross River virus (RRV) are emerging pathogens capable of causing large-scale epidemics of virus-induced arthritis and myositis. The pathology of RRV-induced disease in both humans and mice is associated with induction of the host inflammatory response within the muscle and joints, and prior studies have demonstrated that the host complement system contributes to development of disease. In this study, we have used a mouse model of RRV-induced disease to identify and characterize which complement activation pathways mediate disease progression after infection, and we have identified the mannose binding lectin (MBL) pathway, but not the classical or alternative complement activation pathways, as essential for development of RRV-induced disease. MBL deposition was enhanced in RRV infected muscle tissue from wild type mice and RRV infected MBL deficient mice exhibited reduced disease, tissue damage, and complement deposition compared to wild-type mice. In contrast, mice deficient for key components of the classical or alternative complement activation pathways still developed severe RRV-induced disease. Further characterization of MBL deficient mice demonstrated that similar to C3−/− mice, viral replication and inflammatory cell recruitment were equivalent to wild type animals, suggesting that RRV-mediated induction of complement dependent immune pathology is largely MBL dependent. Consistent with these findings, human patients diagnosed with RRV disease had elevated serum MBL levels compared to healthy controls, and MBL levels in the serum and synovial fluid correlated with severity of disease. These findings demonstrate a role for MBL in promoting RRV-induced disease in both mice and humans and suggest that the MBL pathway of complement activation may be an effective target for therapeutic intervention for humans suffering from RRV-induced arthritis and myositis. PMID:22457620

Ursolic acid mediates photosensitization by initiating mitochondrial-dependent apoptosis

NASA Astrophysics Data System (ADS)

Lee, Yuan-Hao; Wang, Exing; Kumar, Neeru; Glickman, Randolph D.

2013-02-01

The signaling pathways PI3K/Akt and MAPK play key roles in transcription, translation and carcinogenesis, and may be activated by light exposure. These pathways may be modulated or inhibited by naturally-occurring compounds, such as the triterpenoid, ursolic acid (UA). Previously, the transcription factors p53 and NF-kB, which transactivate mitochondrial apoptosis-related genes, were shown to be differentially modulated by UA. Our current work indicates that UA causes these effects via the mTOR and insulin-mediated pathways. UA-modulated apoptosis, following exposure to UV radiation, is observed to correspond to differential levels of oxidative stress in retinal pigment epithelial (RPE) and skin melanoma (SM) cells. Flow cytometry analysis, DHE (dihydroethidium) staining and membrane permeability assay showed that UA pretreatment potentiated cell cycle arrest and radiation-induced apoptosis selectively on SM cells while DNA photo-oxidative damage (i.e. strand breakage) was reduced, presumably by some antioxidant activity of UA in RPE cells. The UA-mediated NF-κB activation in SM cells was reduced by rapamycin pretreatment, which indicates that these agents exert inter-antagonistic effects in the PI3K/Akt/mTOR pathway. In contrast, the antagonistic effect of UA on the PI3K/Akt pathway was reversed by insulin leading to greater NF-κB and p53 activation in RPE cells. MitoTracker, a mitochondrial functional assay, indicated that mitochondria in RPE cells experienced reduced oxidative stress while those in SM cells exhibited increased oxidative stress upon UA pretreatment. When rapamycin administration was followed by UA, mitochondrial oxidative stress was increased in RPE cells but decreased in SM cells. These results indicate that UA modulates p53 and NF-κB, initiating a mitogenic response to radiation that triggers mitochondria-dependent apoptosis.

Overcoming Resistance to Inhibitors of the Akt Protein Kinase by Modulation of the Pim Kinase Pathway

DTIC Science & Technology

2014-10-01

during hypoxia were biologically meaningful, we investigated the activity of a reporter with multiple HIF binding sites ( HRE ) in front of a luciferase...inhibitors in a dose dependent fashion blocked the activity of the HRE to activate luciferase mRNA and protein production. This result demonstrates that

Activation of type 2 cannabinoid receptors (CB2R) promotes fatty acid oxidation through the SIRT1/PGC-1α pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, Xuqin; Sun, Tao; Wang, Xiaodong, E-mail: xdwang666@hotmail.com

2013-07-05

Highlights: •TC, a CB2R specific agonist, stimulates SIRT1 activity by PKA/CREB pathway. •TC promotes PGC-1α transcriptional activity by increasing its deacetylation. •TC increases the expression of genes linked to FAO and promotes the rate of FAO. •The effects of TC in FAO are dependent on CB2R. •Suggesting CB2R as a target to treat diseases with lipid dysregulation. -- Abstract: Abnormal fatty acid oxidation has been associated with obesity and type 2 diabetes. At the transcriptional level, peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α) has been reported to strongly increase the ability of hormone nuclear receptors PPARα and ERRα to drive transcriptionmore » of fatty acid oxidation enzymes. In this study, we report that a specific agonist of the type 2 cannabinoid receptor (CB2R) can lead to fatty acid oxidation through the PGC-1α pathway. We have found that CB2R is expressed in differentiated C2C12 myotubes, and that use of the specific agonist trans-caryophyllene (TC) stimulates sirtuin 1 (SIRT1) deacetylase activity by increasing the phosphorylation of cAMP response element-binding protein (CREB), thus leading to increased levels of PGC-1α deacetylation. This use of TC treatment increases the expression of genes linked to the fatty acid oxidation pathway in a SIRT1/PGC-1α-dependent mechanism and also drastically accelerates the rate of complete fatty acid oxidation in C2C12 myotubes, neither of which occur when CB2R mRNA is knocked down using siRNA. These results reveal that activation of CB2R by a selective agonist promotes lipid oxidation through a signaling/transcriptional pathway. Our findings imply that pharmacological manipulation of CB2R may provide therapeutic possibilities to treat metabolic diseases associated with lipid dysregulation.« less

Mouse Cytotoxic T Cell-derived Granzyme B Activates the Mitochondrial Cell Death Pathway in a Bim-dependent Fashion*

PubMed Central

Catalán, Elena; Jaime-Sánchez, Paula; Aguiló, Nacho; Simon, Markus M.; Froelich, Christopher J.; Pardo, Julián

2015-01-01

Cytotoxic T cells (Tc) use perforin and granzyme B (gzmB) to kill virus-infected cells and cancer cells. Recent evidence suggests that human gzmB primarily induces apoptosis via the intrinsic mitochondrial pathway by either cleaving Bid or activating Bim leading to the activation of Bak/Bax and subsequent generation of active caspase-3. In contrast, mouse gzmB is thought to predominantly induce apoptosis by directly processing pro-caspase-3. However, in certain mouse cell types gzmB-mediated apoptosis mainly occurs via the mitochondrial pathway. To investigate whether Bim is involved under the latter conditions, we have now employed ex vivo virus-immune mouse Tc that selectively kill by using perforin and gzmB (gzmB+Tc) as effector cells and wild type as well as Bim- or Bak/Bax-deficient spontaneously (3T9) or virus-(SV40) transformed mouse embryonic fibroblast cells as targets. We show that gzmB+Tc-mediated apoptosis (phosphatidylserine translocation, mitochondrial depolarization, cytochrome c release, and caspase-3 activation) was severely reduced in 3T9 cells lacking either Bim or both Bak and Bax. This outcome was related to the ability of Tc cells to induce the degradation of Mcl-1 and Bcl-XL, the anti-apoptotic counterparts of Bim. In contrast, gzmB+Tc-mediated apoptosis was not affected in SV40-transformed mouse embryonic fibroblast cells lacking Bak/Bax. The data provide evidence that Bim participates in mouse gzmB+Tc-mediated apoptosis of certain targets by activating the mitochondrial pathway and suggest that the mode of cell death depends on the target cell. Our results suggest that the various molecular events leading to transformation and/or immortalization of cells have an impact on their relative resistance to the multiple gzmB+Tc-induced death pathways. PMID:25605735

Binding Mode Analysis of Zerumbone to Key Signal Proteins in the Tumor Necrosis Factor Pathway

PubMed Central

Fatima, Ayesha; Abdul, Ahmad Bustamam Hj.; Abdullah, Rasedee; Karjiban, Roghayeh Abedi; Lee, Vannajan Sanghiran

2015-01-01

Breast cancer is the second most common cancer among women worldwide. Several signaling pathways have been implicated as causative and progression agents. The tumor necrosis factor (TNF) α protein plays a dual role in promoting and inhibiting cancer depending largely on the pathway initiated by the binding of the protein to its receptor. Zerumbone, an active constituent of Zingiber zerumbet, Smith, is known to act on the tumor necrosis factor pathway upregulating tumour necrosis factor related apoptosis inducing ligand (TRAIL) death receptors and inducing apoptosis in cancer cells. Zerumbone is a sesquiterpene that is able to penetrate into the hydrophobic pockets of proteins to exert its inhibiting activity with several proteins. We found a good binding with the tumor necrosis factor, kinase κB (IKKβ) and the Nuclear factor κB (NF-κB) component proteins along the TNF pathway. Our results suggest that zerumbone can exert its apoptotic activities by inhibiting the cytoplasmic proteins. It inhibits the IKKβ kinase that activates the NF-κB and also binds to the NF-κB complex in the TNF pathway. Blocking both proteins can lead to inhibition of cell proliferating proteins to be downregulated and possibly ultimate induction of apoptosis. PMID:25629232

D4F alleviates macrophage-derived foam cell apoptosis by inhibiting the NF-κB-dependent Fas/FasL pathway.

PubMed

Tian, Hua; Yao, Shu-Tong; Yang, Na-Na; Ren, Jie; Jiao, Peng; Zhang, Xiangjian; Li, Dong-Xuan; Zhang, Gong-An; Xia, Zhen-Fang; Qin, Shu-Cun

2017-08-04

This study was designed to explore the protective effect of D4F, an apolipoprotein A-I mimetic peptide, on nuclear factor-κB (NF-κB)-dependent Fas/Fas ligand (FasL) pathway-mediated apoptosis in macrophages induced by oxidized low-density lipoprotein (ox-LDL). Our results showed that ox-LDL induced apoptosis, NF-κB P65 nuclear translocation and the upregulation of Fas/FasL pathway-related proteins, including Fas, FasL, Fas-associated death domain proteins (FADD), caspase-8 and caspase-3 in RAW264.7 macrophages, whereas silencing of Fas blocked ox-LDL-induced macrophage apoptosis. Furthermore, silencing of P65 attenuated macrophage apoptosis and the upregulation of Fas caused by ox-LDL, whereas P65 expression was not significantly affected by treatment with Fas siRNA. D4F attenuated the reduction of cell viability and the increase in lactate dehydrogenase leakage and apoptosis. Additionally, D4F inhibited ox-LDL-induced P65 nuclear translocation and upregulation of Fas/FasL pathway-related proteins in RAW264.7 cells and in atherosclerotic lesions of apoE -/- mice. However, Jo2, a Fas-activating monoclonal antibody, reversed the inhibitory effect of D4F on ox-LDL-induced cell apoptosis and upregulation of Fas, FasL and FADD. These data indicate that NF-κB mediates Fas/FasL pathway activation and apoptosis in macrophages induced by ox-LDL and that D4F protects macrophages from ox-LDL-induced apoptosis by suppressing the activation of NF-κB and the Fas/FasL pathway.

Poncirin Induces Apoptosis in AGS Human Gastric Cancer Cells through Extrinsic Apoptotic Pathway by up-Regulation of Fas Ligand.

PubMed

Saralamma, Venu Venkatarame Gowda; Nagappan, Arulkumar; Hong, Gyeong Eun; Lee, Ho Jeong; Yumnam, Silvia; Raha, Suchismita; Heo, Jeong Doo; Lee, Sang Joon; Lee, Won Sup; Kim, Eun Hee; Kim, Gon Sup

2015-09-18

Poncirin, a natural bitter flavanone glycoside abundantly present in many species of citrus fruits, has various biological benefits such as anti-oxidant, anti-microbial, anti-inflammatory and anti-cancer activities. The anti-cancer mechanism of Poncirin remains elusive to date. In this study, we investigated the anti-cancer effects of Poncirin in AGS human gastric cancer cells (gastric adenocarcinoma). The results revealed that Poncirin could inhibit the proliferation of AGS cells in a dose-dependent manner. It was observed Poncirin induced accumulation of sub-G1 DNA content, apoptotic cell population, apoptotic bodies, chromatin condensation, and DNA fragmentation in a dose-dependent manner in AGS cells. The expression of Fas Ligand (FasL) protein was up-regulated dose dependently in Poncirin-treated AGS cells Moreover, Poncirin in AGS cells induced activation of Caspase-8 and -3, and subsequent cleavage of poly(ADP-ribose) polymerase (PARP). Inhibitor studies' results confirm that the induction of caspase-dependent apoptotic cell death in Poncirin-treated AGS cells was led by the Fas death receptor. Interestingly, Poncirin did not show any effect on mitochondrial membrane potential (ΔΨm), pro-apoptotic proteins (Bax and Bak) and anti-apoptotic protein (Bcl-xL) in AGS-treated cells followed by no activation in the mitochondrial apoptotic protein caspase-9. This result suggests that the mitochondrial-mediated pathway is not involved in Poncirin-induced cell death in gastric cancer. These findings suggest that Poncirin has a potential anti-cancer effect via extrinsic pathway-mediated apoptosis, possibly making it a strong therapeutic agent for human gastric cancer.

Stimulatory effect of insulin on 5alpha-reductase type 1 (SRD5A1) expression through an Akt-dependent pathway in ovarian granulosa cells.

PubMed

Kayampilly, Pradeep P; Wanamaker, Brett L; Stewart, James A; Wagner, Carrie L; Menon, K M J

2010-10-01

Elevated levels of 5α-reduced androgens have been shown to be associated with hyperandrogenism and hyperinsulinemia, the leading causes of ovulatory dysfunction in women. 5α-Dihydrotestosterone reduces ovarian granulosa cell proliferation by inhibiting FSH-mediated mitogenic signaling pathways. The present study examined the effect of insulin on 5α-reductase, the enzyme that catalyses the conversion of androgens to their 5α-derivatives. Granulosa cells isolated from immature rat ovaries were cultured in serum-free, phenol red-free DMEM-F12 media and treated with different doses of insulin (0, 0.1, 1.0, and 10.0 μg/ml) for different time intervals up to 12 h. The expression of 5α-reductase type 1 mRNA, the predominant isoform found in granulosa cells, showed a significant (P<0.05) increase in response to the insulin treatment up to 12 h compared with control. The catalytic activity of 5α-reductase enzyme was also stimulated in a dose-depended manner (P<0.05). Inhibiting the Akt-dependent signaling pathway abolished the insulin-mediated increase in 5α-reductase mRNA expression, whereas inhibition of the ERK-dependent pathway had no effect. The dose-dependent increase in 5α-reductase mRNA expression as well as catalytic activity seen in response to insulin treatment was also demonstrated in the human granulosa cell line (KGN). In addition to increased mRNA expression, a dose-dependent increase in 5α-reductase protein expression in response to insulin was also seen in KGN cells, which corroborated well with that of mRNA expression. These results suggest that elevated levels of 5α-reduced androgens seen in hyperinsulinemic conditions might be explained on the basis of a stimulatory effect of insulin on 5α-reductase in granulosa cells. The elevated levels of these metabolites, in turn, might adversely affect growth and proliferation of granulosa cells, thereby impairing follicle growth and ovulation.

Inflammation in gastric cancer: Interplay of the COX-2/prostaglandin E2 and Toll-like receptor/MyD88 pathways.

PubMed

Echizen, Kanae; Hirose, Osamu; Maeda, Yusuke; Oshima, Masanobu

2016-04-01

Cyclooxygenase-2 (COX-2) and its downstream product prostaglandin E2 (PGE2 ) play a key role in generation of the inflammatory microenvironment in tumor tissues. Gastric cancer is closely associated with Helicobacter pylori infection, which stimulates innate immune responses through Toll-like receptors (TLRs), inducing COX-2/PGE2 pathway through nuclear factor-κB activation. A pathway analysis of human gastric cancer shows that both the COX-2 pathway and Wnt/β-catenin signaling are significantly activated in tubular-type gastric cancer, and basal levels of these pathways are also increased in other types of gastric cancer. Expression of interleukin-11, chemokine (C-X-C motif) ligand 1 (CXCL1), CXCL2, and CXCL5, which play tumor-promoting roles through a variety of mechanisms, is induced in a COX-2/PGE2 pathway-dependent manner in both human and mouse gastric tumors. Moreover, the COX-2/PGE2 pathway plays an important role in the maintenance of stemness with expression of stem cell markers, including CD44, Prom1, and Sox9, which are induced in both gastritis and gastric tumors through a COX-2/PGE2 -dependent mechanism. In contrast, disruption of Myd88 results in suppression of the inflammatory microenvironment in gastric tumors even when the COX-2/PGE2 pathway is activated, indicating that the interplay of the COX-2/PGE2 and TLR/MyD88 pathways is needed for inflammatory response in tumor tissues. Furthermore, TLR2/MyD88 signaling plays a role in maintenance of stemness in normal stem cells as well as gastric tumor cells. Accordingly, these results suggest that targeting the COX-2/PGE2 pathway together with TLR/MyD88 signaling, which would suppress the inflammatory microenvironment and maintenance of stemness, could be an effective preventive or therapeutic strategy for gastric cancer. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Activation of ERK and JNK signaling pathways by mycotoxin citrinin in human cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, C.-H.; Yu, F.-Y.; Wang, L.-T.

2009-06-15

Mycotoxin citrinin (CTN) is commonly found in foods and feeds that are contaminated/inoculated with Penicillium, Aspergillus and Monascus species. The exposure of human embryonic kidney (HEK293) and HeLa cells to CTN resulted in a dose-dependent increase in the phosphorylation of two major mitogen-activated protein kinases (MAPKs), ERK1/2 and JNK. In HEK293 cultures, the administering of CTN increased both the mRNA and protein levels of egr-1, c-fos and c-jun genes; additionally, the ERK1/2 pathway contributed to the upregulation of Egr-1 and c-Fos protein expression. CTN treatment also induced the transcription activity of Egr-1 and AP-1 proteins, as evidenced by luciferase reportermore » assays. Bioinformatic analyses indicated two genes Gadd45{beta} and MMP3 have Egr-1 and AP-1 response elements in their promoters, respectively. Furthermore, co-exposure of HEK293 cells to CTN and MAPK pathway inhibitors demonstrated that CTN increased the levels of Gadd45{beta} mRNA through ERK1/2 signaling pathway and up-regulated the MMP3 transcripts majorly via JNK pathway. Finally, CTN-triggered caspase 3 activity was significantly reduced in the presence of MAPK inhibitors. Our results suggest that CTN positively regulates ERK1/2 and JNK pathways as well as their downstream effectors in human cells; activated MAPK pathways are also involved in CTN-induced apoptosis.« less

Disruption of Akt kinase activation is important for immunosuppression induced by measles virus.

PubMed

Avota, E; Avots, A; Niewiesk, S; Kane, L P; Bommhardt, U; ter Meulen, V; Schneider-Schaulies, S

2001-06-01

Surface-contact-mediated signaling induced by the measles virus (MV) fusion and hemagglutinin glycoproteins is necessary and sufficient to induce T-cell unresponsiveness in vitro and in vivo. To define the intracellular pathways involved, we analyzed interleukin (IL)-2R signaling in primary human T cells and in Kit-225 cells. Unlike IL-2-dependent activation of JAK/STAT pathways, activation of Akt kinase was impaired after MV contact both in vitro and in vivo. MV interference with Akt activation was important for immunosuppression, as expression of a catalytically active Akt prevented negative signaling by the MV glycoproteins. Thus, we show here that MV exploits a novel strategy to interfere with T-cell activation during immunosuppression.

The Nuclear Receptor AhR Controls Bone Homeostasis by Regulating Osteoclast Differentiation via the RANK/c-Fos Signaling Axis

PubMed Central

Izawa, Takashi; Arakaki, Rieko; Mori, Hiroki; Tsunematsu, Takaaki; Kudo, Yasusei; Tanaka, Eiji

2016-01-01

The aryl hydrocarbon receptor (AhR) pathway plays a key role in receptor activator of NF-κB ligand (RANKL)–mediated osteoclastogenesis. However, the mechanism underlying the regulation of AhR expression in osteoclasts and the signaling pathway through which AhR controls osteoclastogenesis remain unclear. We found that the expression of AhR in bone marrow–derived osteoclasts was upregulated by RANKL at an earlier stage than was the expression of signature osteoclast genes such as those encoding cathepsin K and NFAT, cytoplasmic, calcineurin-dependent 1. In response to RANKL, bone marrow macrophages isolated from AhR−/− mice exhibited impaired phosphorylation of Akt and MAPK as well as NF-κB, whereas their response to M-CSF remained unchanged. Osteoclast differentiation mediated by the AhR signaling pathway was also regulated in an RANKL/c-Fos–dependent manner. Furthermore, ligand activation of AhR by the smoke toxin benzo[a]pyrene accelerated osteoclast differentiation in a receptor-dependent manner, and AhR-dependent regulation of mitochondrial biogenesis in osteoclasts was observed. Moreover, AhR−/− mice exhibited impaired bone healing with delayed endochondral ossification. Taken together, the present results suggest that the RANKL/AhR/c-Fos signaling axis plays a critical role in osteoclastogenesis, thereby identifying the potential of AhR in treating pathological, inflammatory, or metabolic disorders of the bone. PMID:27849171

Quantitative Phospho-proteomic Analysis of TNFα/NFκB Signaling Reveals a Role for RIPK1 Phosphorylation in Suppressing Necrotic Cell Death.

PubMed

Mohideen, Firaz; Paulo, Joao A; Ordureau, Alban; Gygi, Steve P; Harper, J Wade

2017-07-01

TNFα is a potent inducer of inflammation due to its ability to promote gene expression, in part via the NFκB pathway. Moreover, in some contexts, TNFα promotes Caspase-dependent apoptosis or RIPK1/RIPK3/MLKL-dependent necrosis. Engagement of the TNF Receptor Signaling Complex (TNF-RSC), which contains multiple kinase activities, promotes phosphorylation of several downstream components, including TAK1, IKKα/IKKβ, IκBα, and NFκB. However, immediate downstream phosphorylation events occurring in response to TNFα signaling are poorly understood at a proteome-wide level. Here we use Tandem Mass Tagging-based proteomics to quantitatively characterize acute TNFα-mediated alterations in the proteome and phosphoproteome with or without inhibition of the cIAP-dependent survival arm of the pathway with a SMAC mimetic. We identify and quantify over 8,000 phosphorylated peptides, among which are numerous known sites in the TNF-RSC, NFκB, and MAP kinase signaling systems, as well as numerous previously unrecognized phosphorylation events. Functional analysis of S320 phosphorylation in RIPK1 demonstrates a role for this event in suppressing its kinase activity, association with CASPASE-8 and FADD proteins, and subsequent necrotic cell death during inflammatory TNFα stimulation. This study provides a resource for further elucidation of TNFα-dependent signaling pathways. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Iron alters cell survival in a mitochondria-dependent pathway in ovarian cancer cells

PubMed Central

Bauckman, Kyle; Haller, Edward; Taran, Nicholas; Rockfield, Stephanie; Ruiz-Rivera, Abigail; Nanjundan, Meera

2015-01-01

The role of iron in the development of cancer remains unclear. We previously reported that iron reduces cell survival in a Ras/mitogen-activated protein kinase (MAPK)-dependent manner in ovarian cells; however, the underlying downstream pathway leading to reduced survival was unclear. Although levels of intracellular iron, ferritin/CD71 protein and reactive oxygen species did not correlate with iron-induced cell survival changes, we identified mitochondrial damage (via TEM) and reduced expression of outer mitochondrial membrane proteins (translocase of outer membrane: TOM20 and TOM70) in cell lines sensitive to iron. Interestingly, Ru360 (an inhibitor of the mitochondrial calcium uniporter) reversed mitochondrial changes and restored cell survival in HEY ovarian carcinoma cells treated with iron. Further, cells treated with Ru360 and iron also had reduced autophagic punctae with increased lysosomal numbers, implying cross-talk between these compartments. Mitochondrial changes were dependent on activation of the Ras/MAPK pathway since treatment with a MAPK inhibitor restored expression of TOM20/TOM70 proteins. Although glutathione antioxidant levels were reduced in HEY treated with iron, extracellular glutamate levels were unaltered. Strikingly, oxalomalate (inhibitor of aconitase, involved in glutamate production) reversed iron-induced responses in a similar manner to Ru360. Collectively, our results implicate iron in modulating cell survival in a mitochondria-dependent manner in ovarian cancer cells. PMID:25697096

JIP1-Mediated JNK Activation Negatively Regulates Synaptic Plasticity and Spatial Memory.

PubMed

Morel, Caroline; Sherrin, Tessi; Kennedy, Norman J; Forest, Kelly H; Avcioglu Barutcu, Seda; Robles, Michael; Carpenter-Hyland, Ezekiel; Alfulaij, Naghum; Standen, Claire L; Nichols, Robert A; Benveniste, Morris; Davis, Roger J; Todorovic, Cedomir

2018-04-11

The c-Jun N-terminal kinase (JNK) signal transduction pathway is implicated in learning and memory. Here, we examined the role of JNK activation mediated by the JNK-interacting protein 1 (JIP1) scaffold protein. We compared male wild-type mice with a mouse model harboring a point mutation in the Jip1 gene that selectively blocks JIP1-mediated JNK activation. These male mutant mice exhibited increased NMDAR currents, increased NMDAR-mediated gene expression, and a lower threshold for induction of hippocampal long-term potentiation. The JIP1 mutant mice also displayed improved hippocampus-dependent spatial memory and enhanced associative fear conditioning. These results were confirmed using a second JIP1 mutant mouse model that suppresses JNK activity. Together, these observations establish that JIP1-mediated JNK activation contributes to the regulation of hippocampus-dependent, NMDAR-mediated synaptic plasticity and learning. SIGNIFICANCE STATEMENT The results of this study demonstrate that c-Jun N-terminal kinase (JNK) activation induced by the JNK-interacting protein 1 (JIP1) scaffold protein negatively regulates the threshold for induction of long-term synaptic plasticity through the NMDA-type glutamate receptor. This change in plasticity threshold influences learning. Indeed, mice with defects in JIP1-mediated JNK activation display enhanced memory in hippocampus-dependent tasks, such as contextual fear conditioning and Morris water maze, indicating that JIP1-JNK constrains spatial memory. This study identifies JIP1-mediated JNK activation as a novel molecular pathway that negatively regulates NMDAR-dependent synaptic plasticity and memory. Copyright © 2018 the authors 0270-6474/18/383708-21$15.00/0.

Myosin light chain kinase facilitates endocytosis of synaptic vesicles at hippocampal boutons.

PubMed

Li, Lin; Wu, Xiaomei; Yue, Hai-Yuan; Zhu, Yong-Chuan; Xu, Jianhua

2016-07-01

At nerve terminals, endocytosis efficiently recycles vesicle membrane to maintain synaptic transmission under different levels of neuronal activity. Ca(2+) and its downstream signal pathways are critical for the activity-dependent regulation of endocytosis. An activity- and Ca(2+) -dependent kinase, myosin light chain kinase (MLCK) has been reported to regulate vesicle mobilization, vesicle cycling, and motility in different synapses, but whether it has a general contribution to regulation of endocytosis at nerve terminals remains unknown. We investigated this issue at rat hippocampal boutons by imaging vesicle endocytosis as the real-time retrieval of vesicular synaptophysin tagged with a pH-sensitive green fluorescence protein. We found that endocytosis induced by 200 action potentials (5-40 Hz) was slowed by acute inhibition of MLCK and down-regulation of MLCK with RNA interference, while the total amount of vesicle exocytosis and somatic Ca(2+) channel current did not change with MLCK down-regulation. Acute inhibition of myosin II similarly impaired endocytosis. Furthermore, down-regulation of MLCK prevented depolarization-induced phosphorylation of myosin light chain, an effect shared by blockers of Ca(2+) channels and calmodulin. These results suggest that MLCK facilitates vesicle endocytosis through activity-dependent phosphorylation of myosin downstream of Ca(2+) /calmodulin, probably as a widely existing mechanism among synapses. Our study suggests that MLCK is an important activity-dependent regulator of vesicle recycling in hippocampal neurons, which are critical for learning and memory. The kinetics of vesicle membrane endocytosis at nerve terminals has long been known to depend on activity and Ca(2+) . This study provides evidence suggesting that myosin light chain kinase increases endocytosis efficiency at hippocampal neurons by mediating Ca(2+) /calmodulin-dependent phosphorylation of myosin. The authors propose that this signal cascade may serve as a common pathway contributing to the activity-dependent regulation of vesicle endocytosis at synapses. © 2016 International Society for Neurochemistry.

Plasma membrane calcium ATPase isoform 4 inhibits vascular endothelial growth factor-mediated angiogenesis through interaction with calcineurin.

PubMed

Baggott, Rhiannon R; Alfranca, Arantzazu; López-Maderuelo, Dolores; Mohamed, Tamer M A; Escolano, Amelia; Oller, Jorge; Ornes, Beatriz C; Kurusamy, Sathishkumar; Rowther, Farjana B; Brown, James E; Oceandy, Delvac; Cartwright, Elizabeth J; Wang, Weiguang; Gómez-del Arco, Pablo; Martínez-Martínez, Sara; Neyses, Ludwig; Redondo, Juan Miguel; Armesilla, Angel Luis

2014-10-01

Vascular endothelial growth factor (VEGF) has been identified as a crucial regulator of physiological and pathological angiogenesis. Among the intracellular signaling pathways triggered by VEGF, activation of the calcineurin/nuclear factor of activated T cells (NFAT) signaling axis has emerged as a critical mediator of angiogenic processes. We and others previously reported a novel role for the plasma membrane calcium ATPase (PMCA) as an endogenous inhibitor of the calcineurin/NFAT pathway, via interaction with calcineurin, in cardiomyocytes and breast cancer cells. However, the functional significance of the PMCA/calcineurin interaction in endothelial pathophysiology has not been addressed thus far. Using in vitro and in vivo assays, we here demonstrate that the interaction between PMCA4 and calcineurin in VEGF-stimulated endothelial cells leads to downregulation of the calcineurin/NFAT pathway and to a significant reduction in the subsequent expression of the NFAT-dependent, VEGF-activated, proangiogenic genes RCAN1.4 and Cox-2. PMCA4-dependent inhibition of calcineurin signaling translates into a reduction in endothelial cell motility and blood vessel formation that ultimately impairs in vivo angiogenesis by VEGF. Given the importance of the calcineurin/NFAT pathway in the regulation of pathological angiogenesis, targeted modulation of PMCA4 functionality might open novel therapeutic avenues to promote or attenuate new vessel formation in diseases that occur with angiogenesis. © 2014 American Heart Association, Inc.

Role of GPER in estrogen-dependent nitric oxide formation and vasodilation.

PubMed

Fredette, Natalie C; Meyer, Matthias R; Prossnitz, Eric R

2018-02-01

Estrogens are potent regulators of vasomotor tone, yet underlying receptor- and ligand-specific signaling pathways remain poorly characterized. The primary physiological estrogen 17β-estradiol (E2), a non-selective agonist of classical nuclear estrogen receptors (ERα and ERβ) as well as the G protein-coupled estrogen receptor (GPER), stimulates formation of the vasodilator nitric oxide (NO) in endothelial cells. Here, we studied the contribution of GPER signaling in E2-dependent activation of endothelial NO formation and subsequent vasodilation. Employing E2 and the GPER-selective agonist G-1, we investigated eNOS phosphorylation and NO formation in human endothelial cells, and endothelium-dependent vasodilation in the aortae of wild-type and Gper-deficient mice. Both E2 and G-1 induced phosphorylation of eNOS at the activation site Ser1177 to similar extents. Endothelial NO production to E2 was comparable to that of G-1, and was substantially reduced after pharmacological inhibition of GPER. Similarly, the clinically used ER-targeting drugs 4OH-tamoxifen, raloxifene, and ICI182,780 (faslodex, fulvestrant™) induced NO formation in part via GPER. We identified c-Src, EGFR, PI3K and ERK signaling pathways to be involved in GPER-dependent NO formation. In line with activation of NO formation in cells, E2 and G-1 induced equally potent vasodilation in the aorta of wild-type mice. Gper deletion completely abrogated the vasodilator response to G-1, while reducing the response to E2 by ∼50%. These findings indicate that a substantial portion of E2-induced endothelium-dependent vasodilation and NO formation is mediated by GPER. Thus, selective targeting of vascular GPER may be a suitable approach to activate the endothelial NO pathway, possibly leading to reduced vasomotor tone and inhibition of atherosclerotic vascular disease. Copyright © 2017 Elsevier Ltd. All rights reserved.

Progesterone induces progesterone receptor gene (PGR) expression via rapid activation of protein kinase pathways required for cooperative estrogen receptor alpha (ER) and progesterone receptor (PR) genomic action at ER/PR target genes.

PubMed

Diep, Caroline H; Ahrendt, Hannah; Lange, Carol A

2016-10-01

Progesterone Receptors (PRs) are critical effectors of estrogen receptor (ER) signaling required for mammary gland development and reproductive proficiency. In breast and reproductive tract malignancies, PR expression is a clinical prognostic marker of ER action. While estrogens primarily regulate PR expression, other factors likely contribute to a dynamic range of receptor expression across diverse tissues. In this study, we identified estrogen-independent but progestin (R5020)-dependent regulation of ER target genes including PGR in ER+/PR+ cancer cell lines. R5020 (10nM-10μM range) induced dose-dependent PR mRNA and protein expression in the absence of estrogen but required both PR and ERα. Antagonists of either PR (RU486, onapristone) or ERα (ICI 182,780) attenuated R5020 induction of TFF1, CTSD, and PGR. Chromatin immunoprecipitation (ChIP) assays performed on ER+/PR+ cells demonstrated that both ERα and PR were recruited to the same ERE/Sp1 site-containing region of the PGR proximal promoter in response to high dose progestin (10μM). Recruitment of ERα and PR to chromatin and subsequent PR mRNA induction were dependent upon rapid activation of MAPK/ERK and AKT; inhibition of these kinase pathways via U0126 or LY294002 blocked these events. Overall, we have identified a novel mechanism of ERα activation initiated by rapid PR-dependent kinase pathway activation and associated with phosphorylation of ERα Ser118 for estrogen-independent but progestin-dependent ER/PR cross talk. These studies may provide insight into mechanisms of persistent ER-target gene expression during periods of hormone (i.e. estrogen) ablation and suggest caution following prolonged treatment with aromatase or CYP17 inhibitors (i.e. contexts when progesterone levels may be abnormally elevated). Copyright © 2016 Elsevier Inc. All rights reserved.

Academic performance of young competitive swimmers is associated with physical activity intensity and its predominant metabolic pathway: a pilot study.

PubMed

Ayan, Carlos; Cancela Carral, Jose; Montero, Carlos

2014-09-01

The relationship between physical activity (PA) and academic performance has been previously studied. However, there is a need to determine if the intensity of the PA performed and its predominant metabolic pathway show any degree of association with the academic achievement. Cross-sectional data were gathered from Spanish young competitive swimmers. Academic achievement was based on individual grades for each student; the PA level was measured by means of the Physical Activity Questionnaire for Adolescents. Swimmers were classified according to the preferential energetic cost of the event in which they competed. A total of 254 swimmers finished the study; 62.8% of them were considered moderate active. The statistical analysis showed that the higher the level of PA performed, the better the average grades achieved. This relationship was significant among the girls (P = .04). No significant differences were found regarding the influence of the kind of swimming event. However, taking part in aerobic events proved to have a significant influence on the academic achievement for girls (P = .01). The link between academic achievement and PA depends on the intensity in which the PA is performed, as well as on its predominant metabolic pathway. However, such associations seem to be gender-dependent.

The FANC pathway is activated by adenovirus infection and promotes viral replication-dependent recombination

PubMed Central

Cherubini, Gioia; Naim, Valeria; Caruso, Paola; Burla, Romina; Bogliolo, Massimo; Cundari, Enrico; Benihoud, Karim; Saggio, Isabella; Rosselli, Filippo

2011-01-01

Deciphering the crosstalk between a host cell and a virus during infection is important not only to better define viral biology but also to improve our understanding of cellular processes. We identified the FANC pathway as a helper of viral replication and recombination by searching for cellular targets that are modified by adenovirus (Ad) infection and are involved in its outcome. This pathway, which is involved in the DNA damage response and checkpoint control, is altered in Fanconi anaemia, a rare cancer predisposition syndrome. We show here that Ad5 infection activates the FANC pathway independent of the classical DNA damage response. Infection with a non-replicating Ad shows that the presence of viral DNA is not sufficient to induce the monoubiquitination of FANCD2 but still activates the DNA damage response coordinated by phospho-NBS1 and phospho-CHK1. E1A expression alone fails to induce FANCD2 monoubiquitination, indicating that a productive viral infection and/or replication is required for FANC pathway activation. Our data indicate that Ad5 infection induces FANCD2 activation to promote its own replication. Specifically, we show that FANCD2 is involved in the recombination process that accompanies viral DNA replication. This study provides evidence of a DNA damage-independent function of the FANC pathway and identifies a cellular system involved in Ad5 recombination. PMID:21421559

APE1/Ref-1 as an emerging therapeutic target for various human diseases: phytochemical modulation of its functions

PubMed Central

Thakur, Shweta; Sarkar, Bibekananda; Cholia, Ravi P; Gautam, Nandini; Dhiman, Monisha; Mantha, Anil K

2014-01-01

Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional enzyme involved in the base excision repair (BER) pathway, which repairs oxidative base damage caused by endogenous and exogenous agents. APE1 acts as a reductive activator of many transcription factors (TFs) and has also been named redox effector factor 1, Ref-1. For example, APE1 activates activator protein-1, nuclear factor kappa B, hypoxia-inducible factor 1α, paired box gene 8, signal transducer activator of transcription 3 and p53, which are involved in apoptosis, inflammation, angiogenesis and survival pathways. APE1/Ref-1 maintains cellular homeostasis (redox) via the activation of TFs that regulate various physiological processes and that crosstalk with redox balancing agents (for example, thioredoxin, catalase and superoxide dismutase) by controlling levels of reactive oxygen and nitrogen species. The efficiency of APE1/Ref-1's function(s) depends on pairwise interaction with participant protein(s), the functions regulated by APE1/Ref-1 include the BER pathway, TFs, energy metabolism, cytoskeletal elements and stress-dependent responses. Thus, APE1/Ref-1 acts as a ‘hub-protein' that controls pathways that are important for cell survival. In this review, we will discuss APE1/Ref-1's versatile nature in various human etiologies, including neurodegeneration, cancer, cardiovascular and other diseases that have been linked with alterations in the expression, subcellular localization and activities of APE/Ref-1. APE1/Ref-1 can be targeted for therapeutic intervention using natural plant products that modulate the expression and functions of APE1/Ref-1. In addition, studies focusing on translational applications based on APE1/Ref-1-mediated therapeutic interventions are discussed. PMID:25033834

Arsenic-induced cutaneous hyperplastic lesions are associated with the dysregulation of Yap, a Hippo signaling-related protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Changzhao; Srivastava, Ritesh K.; Elmets, Craig A.

2013-09-06

Highlights: •Arsenic activates canonical Hippo signaling pathway and up-regulates αCatenin in the skin. •Arsenic activates transcriptional activity of Yap by its nuclear translocation. •Yap is involved in the disruption of tight/adherens junctions in arsenic-exposed animals. -- Abstract: Arsenic exposure in humans causes a number of toxic manifestations in the skin including cutaneous neoplasm. However, the mechanism of these alterations remains elusive. Here, we provide novel observations that arsenic induced Hippo signaling pathway in the murine skin. This pathway plays crucial roles in determining organ size during the embryonic development and if aberrantly activated in adults, contributes to the pathogenesis ofmore » epithelial neoplasm. Arsenic treatment enhanced phosphorylation-dependent activation of LATS1 kinase and other Hippo signaling regulatory proteins Sav1 and MOB1. Phospho-LATS kinase is known to catalyze the inactivation of a transcriptional co-activator, Yap. However, in arsenic-treated epidermis, we did not observed its inactivation. Thus, as expected, unphosphorylated-Yap was translocated to the nucleus in arsenic-treated epidermis. Yap by binding to the transcription factors TEADs induces transcription of its target genes. Consistently, an up-regulation of Yap-dependent target genes Cyr61, Gli2, Ankrd1 and Ctgf was observed in the skin of arsenic-treated mice. Phosphorylated Yap is important in regulating tight and adherens junctions through its binding to αCatenin. We found disruption of these junctions in the arsenic-treated mouse skin despite an increase in αCatenin. These data provide evidence that arsenic-induced canonical Hippo signaling pathway and Yap-mediated disruption of tight and adherens junctions are independently regulated. These effects together may contribute to the carcinogenic effects of arsenic in the skin.« less

Folic acid inhibits COLO-205 colon cancer cell proliferation through activating the FRα/c-SRC/ERK1/2/NFκB/TP53 pathway: in vitro and in vivo studies

PubMed Central

Kuo, Chun-Ting; Chang, Chieh; Lee, Wen-Sen

2015-01-01

To investigate the molecular mechanism underlying folic acid (FA)-induced anti-colon caner activity, we showed that FA caused G0/G1 arrest in COLO-205. FA activated the proto-oncogene tyrosine-protein kinase Src (c-SRC)-mediated signaling pathway to enhance nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFκB) nuclear translocation and binding onto the tumor protein p53 (TP53) gene promoter, and up-regulated expressions of TP53, cyclin-dependent kinase inhibitor 1A (CDKN1A) and cyclin-dependent kinase inhibitor 1B (CDKN1B). Knock-down of TP53 abolished FA-induced increases in the levels of CDKN1A and CDKN1B protein and G0/G1 arrest in COLO-205. Knock-down of folate receptor alpha (FRα) abolished FA-induced activations in the c-SRC-mediated pathway and increases in the levels of CDKN1A, CDKN1B and TP53 protein. These data suggest that FA inhibited COLO-205 proliferation through activating the FRα/c-SRC/mitogen-activated protein kinase 3/1 (ERK1/2)/NFκB/TP53 pathway-mediated up-regulations of CDKN1A and CDKN1B protein. In vivo studies demonstrated that daily i.p. injections of FA led to profound regression of the COLO-205 tumors and prolong the lifespan. In these tumors, the levels of CDKN1A, CDKN1B and TP53 protein were increased and von willebrand factor (VWF) protein levels were decreased. These findings suggest that FA inhibits COLO-205 colon cancer growth through anti-cancer cell proliferation and anti-angiogenesis. PMID:26056802

Ghrelin augments murine T-cell proliferation by activation of the phosphatidylinositol-3-kinase, extracellular signal-regulated kinase and protein kinase C signaling pathways

PubMed Central

Lee, Jun Ho; Patel, Kalpesh; Tae, Hyun Jin; Lustig, Ana; Kim, Jie Wan; Mattson, Mark P.; Taub, Dennis D.

2014-01-01

Thymic atrophy occurs during normal aging, and is accelerated by exposure to chronic stressors that elevate glucocorticoid levelsand impair the naïve T cell output. The orexigenic hormone ghrelin was recently shown to attenuate age-associated thymic atrophy. Here, we report that ghrelin enhances the proliferation of murine CD4+ primary T cells and a CD4+ T-cell line. Ghrelin induced activation of the ERK1/2 and Akt signaling pathways, via upstream activation of phosphatidylinositol-3-kinase and protein kinase C, to enhance T-cell proliferation. Moreover, ghrelin induced expression of the cell cycle proteins cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2) and retinoblastoma phosphorylation. Finally, ghrelin activated the above-mentioned signaling pathways and stimulated thymocyte proliferation in young and older mice in vivo. PMID:25447526

ERK signaling pathway regulates sleep duration through activity-induced gene expression during wakefulness.

PubMed

Mikhail, Cyril; Vaucher, Angélique; Jimenez, Sonia; Tafti, Mehdi

2017-01-24

Wakefulness is accompanied by experience-dependent synaptic plasticity and an increase in activity-regulated gene transcription. Wake-induced genes are certainly markers of neuronal activity and may also directly regulate the duration of and need for sleep. We stimulated murine cortical cultures with the neuromodulatory signals that are known to control wakefulness in the brain and found that norepinephrine alone or a mixture of these neuromodulators induced activity-regulated gene transcription. Pharmacological inhibition of the various signaling pathways involved in the regulation of gene expression indicated that the extracellular signal-regulated kinase (ERK) pathway is the principal one mediating the effects of waking neuromodulators on gene expression. In mice, ERK phosphorylation in the cortex increased and decreased with wakefulness and sleep. Whole-body or cortical neuron-specific deletion of Erk1 or Erk2 significantly increased the duration of wakefulness in mice, and pharmacological inhibition of ERK phosphorylation decreased sleep duration and increased the duration of wakefulness bouts. Thus, this signaling pathway, which is highly conserved from Drosophila to mammals, is a key pathway that links waking experience-induced neuronal gene expression to sleep duration and quality. Copyright © 2017, American Association for the Advancement of Science.

A high throughput screening for TLR3-IRF3 signaling pathway modulators identifies several antipsychotic drugs as TLR inhibitors1

PubMed Central

Zhu, Jianzhong; Smith, Kevin; Hsieh, Paishiun N.; Mburu, Yvonne K.; Chattopadhyay, Saurabh; Sen, Ganes C.; Sarkar, Saumendra N.

2010-01-01

Toll-like Receptor 3 (TLR3) is one of the major innate immune sensors of double stranded RNA (dsRNA). The signal transduction pathway activated by TLR3, upon binding to dsRNA, leads to the activation of two major transcription factors: NF-κB and IRF3. In an effort to identify specific chemical modulators of TLR3-IRF3 signal transduction pathway we developed a cell-based read out system. Using the interferon stimulated gene 56 (ISG56) promoter driven firefly luciferase gene stably integrated in a TLR3 expressing HEK293 cell line, we were able to generate a cell line where treatment with dsRNA resulted in a dose dependent induction of luciferase activity. A screen of two pharmacologically active compound libraries using this system, identified a number of TLR3-IRF3 signaling pathway modulators. Among them we focused on a subset of inhibitors and characterized their mode of action. Several antipsychotic drugs, such as Sertraline, Trifluoperazine and Fluphenazine were found to be direct inhibitors of the innate immune signaling pathway. These inhibitors also showed the ability to inhibit ISG56 induction mediated by TLR4 and TLR7/8 pathways. Interestingly, they did not show significant effect on TLR3, TLR7 and TLR8 mediated NF-κB activation. Detailed analysis of the signaling pathway indicated that these drugs may be exerting their inhibitory effects on IRF3 via PI3K signaling pathway. The data presented here provides mechanistic explanation of possible anti-inflammatory roles of some antipsychotic drugs. PMID:20382888

Hypoxia triggers a proangiogenic pathway involving cancer cell microvesicles and PAR-2–mediated heparin-binding EGF signaling in endothelial cells

PubMed Central

Svensson, Katrin J.; Kucharzewska, Paulina; Christianson, Helena C.; Sköld, Stefan; Löfstedt, Tobias; Johansson, Maria C.; Mörgelin, Matthias; Bengzon, Johan; Ruf, Wolfram; Belting, Mattias

2011-01-01

Highly malignant tumors, such as glioblastomas, are characterized by hypoxia, endothelial cell (EC) hyperplasia, and hypercoagulation. However, how these phenomena of the tumor microenvironment may be linked at the molecular level during tumor development remains ill-defined. Here, we provide evidence that hypoxia up-regulates protease-activated receptor 2 (PAR-2), i.e., a G-protein–coupled receptor of coagulation-dependent signaling, in ECs. Hypoxic induction of PAR-2 was found to elicit an angiogenic EC phenotype and to specifically up-regulate heparin-binding EGF-like growth factor (HB-EGF). Inhibition of HB-EGF by antibody neutralization or heparin treatment efficiently counteracted PAR-2–mediated activation of hypoxic ECs. We show that PAR-2–dependent HB-EGF induction was associated with increased phosphorylation of ERK1/2, and inhibition of ERK1/2 phosphorylation attenuated PAR-2–dependent HB-EGF induction as well as EC activation. Tissue factor (TF), i.e., the major initiator of coagulation-dependent PAR signaling, was substantially induced by hypoxia in several types of cancer cells, including glioblastoma; however, TF was undetectable in ECs even at prolonged hypoxia, which precludes cell-autonomous PAR-2 activation through TF. Interestingly, hypoxic cancer cells were shown to release substantial amounts of TF that was mainly associated with secreted microvesicles with exosome-like characteristics. Vesicles derived from glioblastoma cells were found to trigger TF/VIIa–dependent activation of hypoxic ECs in a paracrine manner. We provide evidence of a hypoxia-induced signaling axis that links coagulation activation in cancer cells to PAR-2–mediated activation of ECs. The identified pathway may constitute an interesting target for the development of additional strategies to treat aggressive brain tumors. PMID:21788507

Hypoxia triggers a proangiogenic pathway involving cancer cell microvesicles and PAR-2-mediated heparin-binding EGF signaling in endothelial cells.

PubMed

Svensson, Katrin J; Kucharzewska, Paulina; Christianson, Helena C; Sköld, Stefan; Löfstedt, Tobias; Johansson, Maria C; Mörgelin, Matthias; Bengzon, Johan; Ruf, Wolfram; Belting, Mattias

2011-08-09

Highly malignant tumors, such as glioblastomas, are characterized by hypoxia, endothelial cell (EC) hyperplasia, and hypercoagulation. However, how these phenomena of the tumor microenvironment may be linked at the molecular level during tumor development remains ill-defined. Here, we provide evidence that hypoxia up-regulates protease-activated receptor 2 (PAR-2), i.e., a G-protein-coupled receptor of coagulation-dependent signaling, in ECs. Hypoxic induction of PAR-2 was found to elicit an angiogenic EC phenotype and to specifically up-regulate heparin-binding EGF-like growth factor (HB-EGF). Inhibition of HB-EGF by antibody neutralization or heparin treatment efficiently counteracted PAR-2-mediated activation of hypoxic ECs. We show that PAR-2-dependent HB-EGF induction was associated with increased phosphorylation of ERK1/2, and inhibition of ERK1/2 phosphorylation attenuated PAR-2-dependent HB-EGF induction as well as EC activation. Tissue factor (TF), i.e., the major initiator of coagulation-dependent PAR signaling, was substantially induced by hypoxia in several types of cancer cells, including glioblastoma; however, TF was undetectable in ECs even at prolonged hypoxia, which precludes cell-autonomous PAR-2 activation through TF. Interestingly, hypoxic cancer cells were shown to release substantial amounts of TF that was mainly associated with secreted microvesicles with exosome-like characteristics. Vesicles derived from glioblastoma cells were found to trigger TF/VIIa-dependent activation of hypoxic ECs in a paracrine manner. We provide evidence of a hypoxia-induced signaling axis that links coagulation activation in cancer cells to PAR-2-mediated activation of ECs. The identified pathway may constitute an interesting target for the development of additional strategies to treat aggressive brain tumors.

NKT cells in leishmaniasis.

PubMed

Zamora-Chimal, Jaime; Hernández-Ruiz, Joselín; Becker, Ingeborg

2017-04-01

The role of NKT cells in the resistance or susceptibility towards Leishmania infections remains to be defined, since controversial data persist. The response of these cells seems to depend on many variables such as the infection site, the number of infecting parasites, the virulence of the strain and the Leishmania species. We here revise the activation pathways leading to NKT cell activation. NKT cells can be activated by the direct pathway, in which Leishmania glycolipids are presented by CD1d molecules on antigen presenting cells, such as dendritic cells (DC), leading to the secretion of diverse cytokines by NKT. NKT cells can also be activated by the indirect pathway, in which Leishmania glycolipids, such as LPG, stimulate TLR2 in DC, inducing their IL-12 production, which in turn activates NKT cells. The review further analyzes the role of NKT cells in disease development, both in humans as in mouse models. Finally we propose the activation of NKT cells for controlling Leishmania infections. Copyright © 2016 Elsevier GmbH. All rights reserved.

The antiplatelet activity of magnolol is mediated by PPAR-β/γ.

PubMed

Shih, Ching-Yu; Chou, Tz-Chong

2012-09-15

Activation of peroxisome proliferator-activated receptor (PPAR) isoforms (α, β/δ, and γ) is known to inhibit platelet aggregation. In the present study, we examined whether PPARs-mediated pathways contribute to the antiplatelet activity of magnolol, a compound purified from Magnolia officinalis. Magnolol (20-60 μM) dose-dependently enhanced the activity and intracellular level of PPAR-β/γ in platelets. In the presence of selective PPAR-β antagonist (GSK0660) or PPAR-γ antagonist (GW9662), the inhibition of magnolol on collagen-induced platelet aggregation and intracellular Ca(2+) mobilization was significantly reversed. Moreover, magnolol-mediated up-regulation of NO/cyclic GMP/PKG pathway and Akt phosphorylation leading to increase of eNOS activity were markedly abolished by blocking PPAR-β/γ activity. Additionally, magnolol significantly inhibited collagen-induced PKCα activation through a PPAR-β/γ and PKCα interaction manner. The arachidonic acid (AA) or collagen-induced thromboxane B(2) formation and elevation of COX-1 activity caused by AA were also markedly attenuated by magnolol. However, these above effects of magnolol on platelet responses were strongly reduced by simultaneous addition of GSK0660 or GW9662, suggesting that PPAR-β/γ-mediated processes may account for magnolol-regulated antiplatelet mechanisms. Similarly, administration of PPAR-β/γ antagonists remarkably abolished the actions of magnolol in preventing platelet plug formation and prolonging bleeding time in mice. Taken together, we demonstrate for the first time that the antiplatelet and anti-thrombotic activities of magnolol are modulated by up-regulation of PPAR-β/γ-dependent pathways. Copyright © 2012 Elsevier Inc. All rights reserved.

Modelling and simulation of biased agonism dynamics at a G protein-coupled receptor.

PubMed

Bridge, L J; Mead, J; Frattini, E; Winfield, I; Ladds, G

2018-04-07

Theoretical models of G protein-coupled receptor (GPCR) concentration-response relationships often assume an agonist producing a single functional response via a single active state of the receptor. These models have largely been analysed assuming steady-state conditions. There is now much experimental evidence to suggest that many GPCRs can exist in multiple receptor conformations and elicit numerous functional responses, with ligands having the potential to activate different signalling pathways to varying extents-a concept referred to as biased agonism, functional selectivity or pluri-dimensional efficacy. Moreover, recent experimental results indicate a clear possibility for time-dependent bias, whereby an agonist's bias with respect to different pathways may vary dynamically. Efforts towards understanding the implications of temporal bias by characterising and quantifying ligand effects on multiple pathways will clearly be aided by extending current equilibrium binding and biased activation models to include G protein activation dynamics. Here, we present a new model of time-dependent biased agonism, based on ordinary differential equations for multiple cubic ternary complex activation models with G protein cycle dynamics. This model allows simulation and analysis of multi-pathway activation bias dynamics at a single receptor for the first time, at the level of active G protein (α GTP ), towards the analysis of dynamic functional responses. The model is generally applicable to systems with N G G proteins and N* active receptor states. Numerical simulations for N G =N * =2 reveal new insights into the effects of system parameters (including cooperativities, and ligand and receptor concentrations) on bias dynamics, highlighting new phenomena including the dynamic inter-conversion of bias direction. Further, we fit this model to 'wet' experimental data for two competing G proteins (G i and G s ) that become activated upon stimulation of the adenosine A 1 receptor with adenosine derivative compounds. Finally, we show that our model can qualitatively describe the temporal dynamics of this competing G protein activation. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Rosiglitazone attenuates NF-{kappa}B-dependent ICAM-1 and TNF-{alpha} production caused by homocysteine via inhibiting ERK{sub 1/2}/p38MAPK activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bai, Yong-Ping; Liu, Yu-Hui; Chen, Jia

2007-08-17

Previous studies demonstrated an important interaction between nuclear factor-kappaB (NF-{kappa}B) activation and homocysteine (Hcy)-induced cytokines expression in endothelial cells and vascular smooth muscle cells. However, the underlying mechanism remains illusive. In this study, we investigated the effects of Hcy on NF-{kappa}B-mediated sICAM-1, TNF-{alpha} production and the possible involvement of ERK{sub 1/2}/p38MAPK pathway. The effects of rosiglitazone intervention were also examined. Our results show that Hcy increased the levels of sICAM-1 and TNF-{alpha} in cultured human umbilical vein endothelial cells (HUVECs) in a time- and concentration-dependent manner. This effect was significantly depressed by rosiglitazone and different inhibitors (PDTC, NF-{kappa}B inhibitor; PD98059,more » MEK inhibitor; SB203580, p38MAPK specific inhibitor; and staurosporine, PKC inhibitor). Next, we investigated the effect of Hcy on ERK{sub 1/2}/p38MAPK pathway and NF-{kappa}B activity in HUVECs. The results show that Hcy activated both ERK{sub 1/2}/p38MAPK pathway and NF-{kappa}B-DNA-binding activity. These effects were markedly inhibited by rosiglitazone as well as other inhibitors (SB203580, PD98059, and PDTC). Further, the pretreatment of staurosporine abrogated ERK{sub 1/2}/p38MAPK phosphorylation, suggesting that Hcy-induced ERK{sub 1/2}/p38MAPK activation is associated with PKC activity. Our results provide evidence that Hcy-induced NF-{kappa}B activation was mediated by activation of ERK{sub 1/2}/p38MAPK pathway involving PKC activity. Rosiglitazone reduces the NF-{kappa}B-mediated sICAM-1 and TNF-{alpha} production induced by Hcy via inhibition of ERK{sub 1/2}/p38MAPK pa0011thw.« less

Osthole prevents cerebral ischemia-reperfusion injury via the Notch signaling pathway.

PubMed

Guan, Junhong; Wei, Xiangtai; Qu, Shengtao; Lv, Tao; Fu, Qiang; Yuan, Ye

2017-08-01

Stroke is a common cerebrovascular disease in aging populations, and constitutes the second highest principle cause of mortality and the principle cause of permanent disability, and ischemic stroke is the primary form. Osthole is a coumarin derivative extracted from the fruits of Cnidium monnieri (L.) Cusson. In this study, we established a rat model of middle cerebral artery occlusion/reperfusion (MCAO/R) in vivo and found that MCAO/R caused cerebral infarction, hippocampus neuronal injury and apoptosis, and also activated the Notch 1 signaling pathway. However, treatment with osthole further enhanced the activity of Notch 1 signaling and reduced the cerebral infarction as well as the hippocampus neuronal injury and apoptosis induced by MCAO/R in a dose-dependent manner. The same results were observed in a primary neuronal oxygen glucose deficiency/reperfusion (OGD/R) model in vitro, and the effect of osthole could be blocked by an inhibitor of Notch 1 signaling, N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine tert-butyl ester (DAPT). Therefore, we demonstrated that osthole injection prevented rat ischemia-reperfusion injury via activating the Notch 1 signaling pathway in vivo and in vitro in a dose-dependent manner, which may be significant for clinical treatment of ischemic stroke.

Vasopressin up-regulates the expression of growth-related immediate-early genes via two distinct EGF receptor transactivation pathways

PubMed Central

Fuentes, Lida Q.; Reyes, Carlos E.; Sarmiento, José M.; Villanueva, Carolina I.; Figueroa, Carlos D.; Navarro, Javier; González, Carlos B.

2008-01-01

Activation of V1a receptor triggers the expression of growth-related immediate-early genes (IEGs), including c-Fos and Egr-1. Here we found that pre-treatment of rat vascular smooth muscle A-10 cell line with the EGF receptor inhibitor AG1478 or the over-expression of an EGFR dominant negative mutant (HEBCD533) blocked the vasopressin-induced expression of IEGs, suggesting that activation of these early genes mediated by V1a receptor is via transactivation of the EGF receptor. Importantly, the inhibition of the metalloproteinases, which catalyzed the shedding of the EGF receptor agonist HB-EGF, selectively blocked the vasopressin-induced expression c-Fos. On the other hand, the inhibition of c-Src selectively blocked the vasopressin-induced expression of Egr-1. Interestingly, in contrast to the expression of c-Fos, the expression of Egr-1 was mediated via the Ras/MEK/MAPK-dependent signalling pathway. Vasopressin-triggered expression of both genes required the release of intracellular calcium, activation of PKC and β-arrestin 2. These findings demonstrated that vasopressin up-regulated the expression of c-Fos and Erg-1 via transactivation of two distinct EGF receptor-dependent signalling pathways. PMID:18571897

Pheromone-Induced Morphogenesis Improves Osmoadaptation Capacity by Activating the HOG MAPK Pathway**

PubMed Central

Baltanás, Rodrigo; Bush, Alan; Couto, Alicia; Durrieu, Lucía; Hohmann, Stefan; Colman-Lerner, Alejandro

2013-01-01

Environmental and internal conditions expose cells to a multiplicity of stimuli whose consequences are difficult to predict. Here, we investigate the response to mating pheromone of yeast cells adapted to high osmolarity. Events downstream of pheromone binding involve two mitogen-activated protein kinase (MAPK) cascades: the pheromone response (PR) and the cell-wall integrity response (CWI). Although these MAPK pathways share components with each and a third MAPK pathway, the high osmolarity response (HOG), they are normally only activated by distinct stimuli, a phenomenon called insulation. We found that in cells adapted to high osmolarity, PR activated the HOG pathway in a pheromone- and osmolarity- dependent manner. Activation of HOG by the PR was not due to loss of insulation, but rather a response to a reduction in internal osmolarity, which resulted from an increase in glycerol release caused by the PR. By analyzing single-cell time courses, we found that stimulation of HOG occurred in discrete bursts that coincided with the “shmooing” morphogenetic process. Activation required the polarisome, the cell wall integrity MAPK Slt2, and the aquaglyceroporin Fps1. HOG activation resulted in high glycerol turnover that improved adaptability to rapid changes in osmolarity. Our work shows how a differentiation signal can recruit a second, unrelated sensory pathway to enable responses to yeast to multiple stimuli. PMID:23612707

Malignant transformation of CD4+ T lymphocytes mediated by oncogenic kinase NPM/ALK recapitulates IL-2-induced cell signaling and gene expression reprogramming

PubMed Central

Marzec, Michal; Halasa, Krzysztof; Liu, Xiaobin; Wang, Hong Y.; Cheng, Mangeng; Baldwin, Donald; Tobias, John W.; Schuster, Stephen J.; Woetmann, Anders; Zhang, Qian; Turner, Suzanne D.; Odum, Niels; Wasik, Mariusz A.

2013-01-01

Anaplastic lymphoma kinase (ALK) physiologically expressed only by nervous system cells displays remarkable capacity to transform CD4+ T lymphocytes and other types of non-neural cells. Here we report that activity of nucleophosphmin (NPM)/ALK chimeric protein, the dominant form of ALK expressed in T-cell lymphomas (ALK+TCL), closely resembles cell activation induced by interleukin 2 (IL-2), the key cytokine supporting growth and survival of normal CD4+ T lymphocytes. Direct comparison of gene expression by ALK+TCL cells treated with an ALK inhibitor and IL-2-dependent ALK-TCL cells stimulated with the cytokine revealed a very similar, albeit inverse, gene regulation pattern. Depending on the analysis method, up to 67% of the modulated genes could be defined as modulated in common by NPM/ALK and IL-2. Based on the gene expression patterns, Jak/STAT and IL-2 signaling pathways topped the list of pathways identified as affected by both IL-2 and NPM/ALK. The expression dependence on NPM/ALK and IL-2 of the five selected genes: CD25 (IL-2Rα), Egr-1, Fosl-1, SOCS3, and Irf-4 was confirmed at the protein level. In both ALK+TCL and IL-2-stimulated ALK-TCL cells, CD25, SOCS3, and Irf-4 genes were activated predominantly by the STAT5 and STAT3 transcription factors, while transcription of Egr-1 and Fosl-1 was induced by the MEK-ERK pathway. Finally, we found that Egr-1, a protein not associated previously with either IL-2 or ALK, contributes to the cell proliferation. These findings indicate that NPM/ALK transforms the target CD4+ T lymphocytes, at least in part, by utilizing the pre-existing, IL-2-dependent signaling pathways. PMID:24218456

N-(3-Oxo-acyl)-homoserine lactone induces apoptosis primarily through a mitochondrial pathway in fibroblasts.

PubMed

Neely, Aaron M; Zhao, Guoping; Schwarzer, Christian; Stivers, Nicole S; Whitt, Aaron G; Meng, Shuhan; Burlison, Joseph A; Machen, Terry E; Li, Chi

2018-01-01

N-(3-Oxododecanoyl)-l-homoserine lactone (C12) is produced by Pseudomonas aeruginosa to function as a quorum-sensing molecule for bacteria-bacteria communication. C12 is also known to influence many aspects of human host cell physiology, including induction of cell death. However, the signalling pathway(s) leading to C12-triggered cell death is (are) still not completely known. To clarify cell death signalling induced by C12, we examined mouse embryonic fibroblasts deficient in "initiator" caspases or "effector" caspases. Our data indicate that C12 selectively induces the mitochondria-dependent intrinsic apoptotic pathway by quickly triggering mitochondrial outer membrane permeabilisation. Importantly, the activities of C12 to permeabilise mitochondria are independent of activation of both "initiator" and "effector" caspases. Furthermore, C12 directly induces mitochondrial outer membrane permeabilisation in vitro. Overall, our study suggests a mitochondrial apoptotic signalling pathway triggered by C12, in which C12 or its metabolite(s) acts on mitochondria to permeabilise mitochondria, leading to activation of apoptosis. © 2017 John Wiley & Sons Ltd.

Hippo signaling pathway in liver and pancreas: the potential drug target for tumor therapy.

PubMed

Kong, Delin; Zhao, Yicheng; Men, Tong; Teng, Chun-Bo

2015-02-01

Cell behaviors, including proliferation, differentiation and apoptosis, are intricately controlled during organ development and tissue regeneration. In the past 9 years, the Hippo signaling pathway has been delineated to play critical roles in organ size control, tissue regeneration and tumorigenesis through regulating cell behaviors. In mammals, the core modules of the Hippo signaling pathway include the MST1/2-LATS1/2 kinase cascade and the transcriptional co-activators YAP/TAZ. The activity of YAP/TAZ is suppressed by cytoplasmic retention due to phosphorylation in the canonical MST1/2-LATS1/2 kinase cascade-dependent manner or the non-canonical MST1/2- and/or LATS1/2-independent manner. Hippo signaling pathway, which can be activated or inactivated by cell polarity, contact inhibition, mechanical stretch and extracellular factors, has been demonstrated to be involved in development and tumorigenesis of liver and pancreas. In addition, we have summarized several small molecules currently available that can target Hippo-YAP pathway for potential treatment of hepatic and pancreatic cancers, providing clues for other YAP initiated cancers therapy as well.

Activation of DNA damage repair pathways by murine polyomavirus.

PubMed

Heiser, Katie; Nicholas, Catherine; Garcea, Robert L

2016-10-01

Nuclear replication of DNA viruses activates DNA damage repair (DDR) pathways, which are thought to detect and inhibit viral replication. However, many DNA viruses also depend on these pathways in order to optimally replicate their genomes. We investigated the relationship between murine polyomavirus (MuPyV) and components of DDR signaling pathways including CHK1, CHK2, H2AX, ATR, and DNAPK. We found that recruitment and retention of DDR proteins at viral replication centers was independent of H2AX, as well as the viral small and middle T-antigens. Additionally, infectious virus production required ATR kinase activity, but was independent of CHK1, CHK2, or DNAPK signaling. ATR inhibition did not reduce the total amount of viral DNA accumulated, but affected the amount of virus produced, indicating a defect in virus assembly. These results suggest that MuPyV may utilize a subset of DDR proteins or non-canonical DDR signaling pathways in order to efficiently replicate and assemble. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Cot/tpl2 activity is required for TLR-induced activation of the Akt p70 S6k pathway in macrophages: Implications for NO synthase 2 expression.

PubMed

López-Peláez, Marta; Soria-Castro, Irene; Boscá, Lisardo; Fernández, Margarita; Alemany, Susana

2011-06-01

LPS stimulation activates IKK and different MAP kinase pathways, as well as the PI3K-Akt-mTOR-p70 S6k pathway, a negative regulator of these MyD88-dependent intracellular signals. Here, we show that Cot/tpl2, a MAP3K responsible for the activation of the MKK1-Erk1/2, controls P-Ser473 Akt and P-Thr389 p70 S6k phosphorylation in LPS-stimulated macrophages. Analysis of the intracellular signalling in Cot/tpl2 KO macrophages versus WT macrophages reveals lower IκBα recovery and higher phosphorylation of JNK and p38α after 1 h of LPS stimulation. Moreover, Cot/tpl2 deficiency increases LPS-induced NO synthase 2 (NOS2) expression in macrophages. Inhibition of the PI3K pathway abolishes the differences in IκBα and NOS2 expression between Cot/tpl2 KO and WT macrophages following LPS administration. Furthermore, in zymosan- and polyI:C-stimulated macrophages, Cot/tpl2 mediates P-Ser473 Akt phosphorylation, increases IκBα levels and decreases NOS2 expression. In conclusion, these data reveal a novel role for the Cot/tpl2 pathway in mediating TLR activation of the Akt-mTOR-p70 S6k pathway, allowing Cot/tpl2 to fine-control the activation state of other signalling pathways. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Curcumin inhibits the invasion of lung cancer cells by modulating the PKCα/Nox-2/ROS/ATF-2/MMP-9 signaling pathway.

PubMed

Fan, Zhigang; Duan, Xiaoyi; Cai, Hui; Wang, Li; Li, Min; Qu, Jingkun; Li, Wanjun; Wang, Yongheng; Wang, Jiansheng

2015-08-01

Invasion and metastasis are the major causes of tumor-related mortality in lung cancer. It is believed that curcumin is an effective drug possessing anti-invasive and anti-metastatic activities in the treatment of cancer. However, the specific mechanisms remain unclear. In the present study, we investigated whether the PKCα/Nox-2/ATF-2/MMP-9 signaling pathway is involved in the invasive behavior of lung cancer and whether curcumin could inhibit invasion by modulating this pathway. The cytotoxic effect of curcumin was evaluated by MTT assay and the capacity of invasion was assessed by Transwell assay. siRNA and plasmid transfection techniques were used to study the function of targeted genes. Real-time PCR and western blot analysis were used to evaluate the expression levels of PKCα, Nox-2, MMP-9 and the phosphorylation of ATF-2. The results showed that curcumin inhibited the proliferation and invasion of A549 cells in a dose-dependent manner. Overexpression of MMP-9 enhanced the invasion of A549 cells. However, inhibition of MMP-9 by siRNA or curcumin suppressed cell invasion. Moreover, we also demonstrated the catalytic role of PKCα in expression of MMP-9 and cellular invasion in A549 cells, which was dependent on the expression of Nox-2 and phosphorylation of ATF-2. Finally, we also showed that curcumin dose-dependently reduced the expression of PKCα, P47phox, Nox-2 and phosphorylated ATF-2, as well as intracellular ROS generation, suggesting the inhibitory effect of curcumin on the activation of the PKCα/Nox-2/ROS/ATF-2 pathway. In conclusion, the PKCα/Nox-2/ROS/ATF-2/MMP-9 signaling pathway is activated in lung cancer A549 cells, which could be modulated by curcumin to inhibit cell invasiveness.

Design and Functional Characterization of a Novel, Arrestin-Biased Designer G Protein-Coupled Receptor

PubMed Central

Nakajima, Ken-ichiro

2012-01-01

Mutational modification of distinct muscarinic receptor subtypes has yielded novel designer G protein-coupled receptors (GPCRs) that are unable to bind acetylcholine (ACh), the endogenous muscarinic receptor ligand, but can be efficiently activated by clozapine-N-oxide (CNO), an otherwise pharmacologically inert compound. These CNO-sensitive designer GPCRs [alternative name: designer receptors exclusively activated by designer drug (DREADDs)] have emerged as powerful new tools to dissect the in vivo roles of distinct G protein signaling pathways in specific cell types or tissues. As is the case with other GPCRs, CNO-activated DREADDs not only couple to heterotrimeric G proteins but can also recruit proteins of the arrestin family (arrestin-2 and -3). Accumulating evidence suggests that arrestins can act as scaffolding proteins to promote signaling through G protein-independent signaling pathways. To explore the physiological relevance of these arrestin-dependent signaling pathways, the availability of an arrestin-biased DREADD would be highly desirable. In this study, we describe the development of an M3 muscarinic receptor-based DREADD [Rq(R165L)] that is no longer able to couple to G proteins but can recruit arrestins and promote extracellular signal-regulated kinase-1/2 phosphorylation in an arrestin- and CNO-dependent fashion. Moreover, CNO treatment of mouse insulinoma (MIN6) cells expressing the Rq(R165L) construct resulted in a robust, arrestin-dependent stimulation of insulin release, directly implicating arrestin signaling in the regulation of insulin secretion. This newly developed arrestin-biased DREADD represents an excellent novel tool to explore the physiological relevance of arrestin signaling pathways in distinct tissues and cell types. PMID:22821234

Sigmar1 regulates endoplasmic reticulum stress-induced C/EBP-homologous protein expression in cardiomyocytes.

PubMed

Alam, Shafiul; Abdullah, Chowdhury S; Aishwarya, Richa; Orr, A Wayne; Traylor, James; Miriyala, Sumitra; Panchatcharam, Manikandan; Pattillo, Christopher B; Bhuiyan, Md Shenuarin

2017-08-31

C/EBP-homologous protein (CHOP) is a ubiquitously expressed stress-inducible transcription factor robustly induced by maladaptive endoplasmic reticulum (ER) stresses in a wide variety of cells. Here, we examined a novel function of Sigma 1 receptor (Sigmar1) in regulating CHOP expression under ER stress in cardiomyocytes. We also defined Sigmar1-dependent activation of the adaptive ER-stress pathway in regulating CHOP expression. We used adenovirus-mediated Sigmar1 overexpression as well as Sigmar1 knockdown by siRNA in neonatal rat ventricular cardiomyocytes (NRCs); to induce ER stress, cardiomyocytes were treated with tunicamycin. Sigmar1-siRNA knockdown significantly increased the expression of CHOP and significantly induced cellular toxicity by sustained activation of ER stress in cardiomyocytes. Sigmar1 overexpression decreased the expression of CHOP and significantly decreased cellular toxicity in cells. Using biochemical and immunocytochemical experiments, we also defined the specific ER-stress pathway associated with Sigmar1-dependent regulation of CHOP expression and cellular toxicity. We found that Sigmar1 overexpression significantly increased inositol requiring kinase 1α (IRE1α) phosphorylation and increased spliced X-box-binding proteins (XBP1s) expression as well as nuclear localization. In contrast, Sigmar1 knockdown significantly decreased IRE1α phosphorylation and decreased XBP1s expression as well as nuclear transport. Taken together, these results indicate that Sigmar1-dependent activation of IRE1α-XBP1s ER-stress response pathways are associated with inhibition of CHOP expression and suppression of cellular toxicity. Hence, Sigmar1 is an essential component of the adaptive ER-stress response pathways eliciting cellular protection in cardiomyocytes. © 2017 The Author(s).

Sigmar1 regulates endoplasmic reticulum stress-induced C/EBP-homologous protein expression in cardiomyocytes

PubMed Central

Alam, Shafiul; Abdullah, Chowdhury S.; Aishwarya, Richa; Orr, A. Wayne; Traylor, James; Miriyala, Sumitra; Panchatcharam, Manikandan; Pattillo, Christopher B.

2017-01-01

C/EBP-homologous protein (CHOP) is a ubiquitously expressed stress-inducible transcription factor robustly induced by maladaptive endoplasmic reticulum (ER) stresses in a wide variety of cells. Here, we examined a novel function of Sigma 1 receptor (Sigmar1) in regulating CHOP expression under ER stress in cardiomyocytes. We also defined Sigmar1-dependent activation of the adaptive ER-stress pathway in regulating CHOP expression. We used adenovirus-mediated Sigmar1 overexpression as well as Sigmar1 knockdown by siRNA in neonatal rat ventricular cardiomyocytes (NRCs); to induce ER stress, cardiomyocytes were treated with tunicamycin. Sigmar1-siRNA knockdown significantly increased the expression of CHOP and significantly induced cellular toxicity by sustained activation of ER stress in cardiomyocytes. Sigmar1 overexpression decreased the expression of CHOP and significantly decreased cellular toxicity in cells. Using biochemical and immunocytochemical experiments, we also defined the specific ER-stress pathway associated with Sigmar1-dependent regulation of CHOP expression and cellular toxicity. We found that Sigmar1 overexpression significantly increased inositol requiring kinase 1α (IRE1α) phosphorylation and increased spliced X-box-binding proteins (XBP1s) expression as well as nuclear localization. In contrast, Sigmar1 knockdown significantly decreased IRE1α phosphorylation and decreased XBP1s expression as well as nuclear transport. Taken together, these results indicate that Sigmar1-dependent activation of IRE1α-XBP1s ER-stress response pathways are associated with inhibition of CHOP expression and suppression of cellular toxicity. Hence, Sigmar1 is an essential component of the adaptive ER-stress response pathways eliciting cellular protection in cardiomyocytes. PMID:28667101

Signal persistence and amplification in cancer development and possible, related opportunities for novel therapies.

PubMed

Ford, Shea A; Blanck, George

2015-01-01

Research in cancer biology has been largely driven by experimental approaches whereby discreet inputs are used to assess discreet outputs, for example, gene-knockouts to assess cancer occurrence. However, cancer hallmarks are only rarely, if ever, exclusively dependent on discreet regulatory processes. Rather, cancer-related regulatory factors affect multiple cancer hallmarks. Thus, novel approaches and paradigms are needed for further advances. Signal pathway persistence and amplification, rather than signal pathway activation resulting from an on/off switch, represent emerging paradigms for cancer research, closely related to developmental regulatory paradigms. In this review, we address both mechanisms and effects of signal pathway persistence and amplification in cancer settings; and address the possibility that hyper-activation of pro-proliferative signal pathways in certain cancer settings could be exploited for therapy. Copyright © 2014. Published by Elsevier B.V.

The MST/Hippo Pathway and Cell Death: A Non-Canonical Affair

PubMed Central

Fallahi, Emma; O’Driscoll, Niamh A.; Matallanas, David

2016-01-01

The MST/Hippo signalling pathway was first described over a decade ago in Drosophila melanogaster and the core of the pathway is evolutionary conserved in mammals. The mammalian MST/Hippo pathway regulates organ size, cell proliferation and cell death. In addition, it has been shown to play a central role in the regulation of cellular homeostasis and it is commonly deregulated in human tumours. The delineation of the canonical pathway resembles the behaviour of the Hippo pathway in the fly where the activation of the core kinases of the pathway prevents the proliferative signal mediated by the key effector of the pathway YAP. Nevertheless, several lines of evidence support the idea that the mammalian MST/Hippo pathway has acquired new features during evolution, including different regulators and effectors, crosstalk with other essential signalling pathways involved in cellular homeostasis and the ability to actively trigger cell death. Here we describe the current knowledge of the mechanisms that mediate MST/Hippo dependent cell death, especially apoptosis. We include evidence for the existence of complex signalling networks where the core proteins of the pathway play a central role in controlling the balance between survival and cell death. Finally, we discuss the possible involvement of these signalling networks in several human diseases such as cancer, diabetes and neurodegenerative disorders. PMID:27322327

Excitation-transcription coupling in parvalbumin-positive interneurons employs a novel CaM Kinase-dependent pathway distinct from excitatory neurons

PubMed Central

Cohen, Samuel M.; Ma, Huan; Kuchibhotla, Kishore V.; Watson, Brendon O.; Buzsáki, György; Froemke, Robert C.; Tsien, Richard W.

2016-01-01

Properly functional CNS circuits depend on inhibitory interneurons that in turn rely upon activity-dependent gene expression for morphological development, connectivity and excitatory-inhibitory coordination. Despite its importance, excitation-transcription coupling in inhibitory interneurons is poorly understood. Here, we report that PV+ interneurons employ a novel CaMK-dependent pathway to trigger CREB phosphorylation and gene expression. As in excitatory neurons, voltage-gated Ca2+ influx through CaV1 channels triggers CaM nuclear translocation via local Ca2+ signaling. However, PV+ interneurons are distinct in that nuclear signaling is mediated by γCaMKI, not γCaMKII. CREB phosphorylation also proceeds with slow, sigmoid kinetics, rate-limited by paucity of CaMKIV, protecting against saturation of phospho-CREB in the face of higher firing rates and bigger Ca2+ transients. Our findings support the generality of CaM shuttling to drive nuclear CaMK activity, and are relevant to disease pathophysiology, insofar as dysfunction of PV+ interneurons and molecules underpinning their excitation-transcription coupling both relate to neuropsychiatric disease. PMID:27041500

Spermidine promotes stress resistance in Drosophila melanogaster through autophagy-dependent and -independent pathways.

PubMed

Minois, N; Carmona-Gutierrez, D; Bauer, M A; Rockenfeller, P; Eisenberg, T; Brandhorst, S; Sigrist, S J; Kroemer, G; Madeo, F

2012-10-11

The naturally occurring polyamine spermidine (Spd) has recently been shown to promote longevity across species in an autophagy-dependent manner. Here, we demonstrate that Spd improves both survival and locomotor activity of the fruit fly Drosophila melanogaster upon exposure to the superoxide generator and neurotoxic agent paraquat. Although survival to a high paraquat concentration (20 mM) was specifically increased in female flies only, locomotor activity and survival could be rescued in both male and female animals when exposed to lower paraquat levels (5 mM). These effects are dependent on the autophagic machinery, as Spd failed to confer resistance to paraquat-induced toxicity and locomotor impairment in flies deleted for the essential autophagic regulator ATG7 (autophagy-related gene 7). Spd treatment did also protect against mild doses of another oxidative stressor, hydrogen peroxide, but in this case in an autophagy-independent manner. Altogether, this study establishes that the protective effects of Spd can be exerted through different pathways that depending on the oxidative stress scenario do or do not involve autophagy.

Lipid-induced insulin resistance mediated by the proinflammatory receptor TLR4 requires saturated fatty acid-induced ceramide biosynthesis in mice.

PubMed

Holland, William L; Bikman, Benjamin T; Wang, Li-Ping; Yuguang, Guan; Sargent, Katherine M; Bulchand, Sarada; Knotts, Trina A; Shui, Guanghou; Clegg, Deborah J; Wenk, Markus R; Pagliassotti, Michael J; Scherer, Philipp E; Summers, Scott A

2011-05-01

Obesity is associated with an enhanced inflammatory response that exacerbates insulin resistance and contributes to diabetes, atherosclerosis, and cardiovascular disease. One mechanism accounting for the increased inflammation associated with obesity is activation of the innate immune signaling pathway triggered by TLR4 recognition of saturated fatty acids, an event that is essential for lipid-induced insulin resistance. Using in vitro and in vivo systems to model lipid induction of TLR4-dependent inflammatory events in rodents, we show here that TLR4 is an upstream signaling component required for saturated fatty acid-induced ceramide biosynthesis. This increase in ceramide production was associated with the upregulation of genes driving ceramide biosynthesis, an event dependent of the activity of the proinflammatory kinase IKKβ. Importantly, increased ceramide production was not required for TLR4-dependent induction of inflammatory cytokines, but it was essential for TLR4-dependent insulin resistance. These findings suggest that sphingolipids such as ceramide might be key components of the signaling networks that link lipid-induced inflammatory pathways to the antagonism of insulin action that contributes to diabetes.

Glycoprotein Ib activation by thrombin stimulates the energy metabolism in human platelets

PubMed Central

Corona de la Peña, Norma; Gutiérrez-Aguilar, Manuel; Hernández-Reséndiz, Ileana; Marín-Hernández, Álvaro

2017-01-01

Thrombin-induced platelet activation requires substantial amounts of ATP. However, the specific contribution of each ATP-generating pathway i.e., oxidative phosphorylation (OxPhos) versus glycolysis and the biochemical mechanisms involved in the thrombin-induced activation of energy metabolism remain unclear. Here we report an integral analysis on the role of both energy pathways in human platelets activated by several agonists, and the signal transducing mechanisms associated with such activation. We found that thrombin, Trap-6, arachidonic acid, collagen, A23187, epinephrine and ADP significantly increased glycolytic flux (3–38 times vs. non-activated platelets) whereas ristocetin was ineffective. OxPhos (33 times) and mitochondrial transmembrane potential (88%) were increased only by thrombin. OxPhos was the main source of ATP in thrombin-activated platelets, whereas in platelets activated by any of the other agonists, glycolysis was the principal ATP supplier. In order to establish the biochemical mechanisms involved in the thrombin-induced OxPhos activation in platelets, several signaling pathways associated with mitochondrial activation were analyzed. Wortmannin and LY294002 (PI3K/Akt pathway inhibitors), ristocetin and heparin (GPIb inhibitors) as well as resveratrol, ATP (calcium-release inhibitors) and PP1 (Tyr-phosphorylation inhibitor) prevented the thrombin-induced platelet activation. These results suggest that thrombin activates OxPhos and glycolysis through GPIb-dependent signaling involving PI3K and Akt activation, calcium mobilization and protein phosphorylation. PMID:28817667

TCDD and omeprazole prime platelets through the aryl hydrocarbon receptor (AhR) non-genomic pathway.

PubMed

Pombo, Mónica; Lamé, Michael W; Walker, Naomi J; Huynh, Danh H; Tablin, Fern

2015-05-19

The role of the aryl hydrocarbon receptor (AhR) in hemostasis has recently gained increased attention. Here, we demonstrate, by qRT-PCR and western blot, that human platelets express both AhR mRNA and AhR protein. AhR protein levels increase in a dose dependent manner when incubated with either 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or omeprazole. Treatment of platelets with puromycin blocks increased AhR protein synthesis in the presence of AhR activators. Additionally, treatment of platelets with either activator results in phosphorylation of p38MAPK and cPLA2, two key signaling molecules in platelet activation pathways. Using the AhR competitive inhibitors alpha naphthoflavone and CH-223191, we show that phosphorylation of p38MAPK is AhR dependent. Further, inhibition of p38MAPK blocks downstream cPLA2 phosphorylation induced by TCDD or omeprazole. Treatment with AhR activators results in platelet priming, as demonstrated by increased platelet aggregation, which is inhibited by AhR antagonists. Our data support a model of the platelet AhR non-genomic pathway in which treatment with AhR activators results in increased expression of the AhR, phosphorylation of p38MAPK and cPLA2, leading to platelet priming in response to agonist. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Single-cell analysis of dihydroartemisinin-induced apoptosis through reactive oxygen species-mediated caspase-8 activation and mitochondrial pathway in ASTC-a-1 cells using fluorescence imaging techniques

NASA Astrophysics Data System (ADS)

Lu, Ying-Ying; Chen, Tong-Sheng; Wang, Xiao-Ping; Li, Li

2010-07-01

Dihydroartemisinin (DHA), a front-line antimalarial herbal compound, has been shown to possess promising anticancer activity with low toxicity. We have previously reported that DHA induced caspase-3-dependent apoptosis in human lung adenocarcinoma cells. However, the cellular target and molecular mechanism of DHA-induced apoptosis is still poorly defined. We use confocal fluorescence microscopy imaging, fluorescence resonance energy transfer, and fluorescence recovery after photobleaching techniques to explore the roles of DHA-elicited reactive oxygen species (ROS) in the DHA-induced Bcl-2 family proteins activation, mitochondrial dysfunction, caspase cascade, and cell death. Cell Counting Kit-8 assay and flow cytometry analysis showed that DHA induced ROS-mediated apoptosis. Confocal imaging analysis in a single living cell and Western blot assay showed that DHA triggered ROS-dependent Bax translocation, mitochondrial membrane depolarization, alteration of mitochondrial morphology, cytochrome c release, caspase-9, caspase-8, and caspase-3 activation, indicating the coexistence of ROS-mediated mitochondrial and death receptor pathway. Collectively, our findings demonstrate for the first time that DHA induces cell apoptosis by triggering ROS-mediated caspase-8/Bid activation and the mitochondrial pathway, which provides some novel insights into the application of DHA as a potential anticancer drug and a new therapeutic strategy by targeting ROS signaling in lung adenocarcinoma therapy in the future.

Nuclear-Localized and Deregulated Calcium- and Calmodulin-Dependent Protein Kinase Activates Rhizobial and Mycorrhizal Responses in Lotus japonicus[W

PubMed Central

Takeda, Naoya; Maekawa, Takaki; Hayashi, Makoto

2012-01-01

The common symbiosis pathway is at the core of symbiosis signaling between plants and soil microbes. In this pathway, calcium- and calmodulin-dependent protein kinase (CCaMK) plays a crucial role in integrating the signals both in arbuscular mycorrhizal symbiosis (AMS) and in root nodule symbiosis (RNS). However, the molecular mechanism by which CCaMK coordinates AMS and RNS is largely unknown. Here, we report that the gain-of-function (GOF) variants of CCaMK without the regulatory domains activate both AMS and RNS signaling pathways in the absence of symbiotic partners. This activation requires nuclear localization of CCaMK. Enforced nuclear localization of the GOF-CCaMK variants by fusion with a canonical nuclear localization signal enhances signaling activity of AMS and RNS. The GOF-CCaMK variant triggers formation of a structure similar to the prepenetration apparatus, which guides infection of arbuscular mycorrhizal fungi to host root cells. In addition, the GOF-CCaMK variants without the regulatory domains partly restore AMS but fail to support rhizobial infection in ccamk mutants. These data indicate that AMS, the more ancient type of symbiosis, can be mainly regulated by the kinase activity of CCaMK, whereas RNS, which evolved more recently, requires complex regulation performed by the regulatory domains of CCaMK. PMID:22337918

Activated AKT/PKB signaling in C. elegans uncouples temporally distinct outputs of DAF-2/insulin-like signaling

PubMed Central

Gami, Minaxi S; Iser, Wendy B; Hanselman, Keaton B; Wolkow, Catherine A

2006-01-01

Background In the nematode, Caenorhabditis elegans, a conserved insulin-like signaling pathway controls larval development, stress resistance and adult lifespan. AGE-1, a homolog of the p110 catalytic subunit of phosphoinositide 3-kinases (PI3K) comprises the major known effector pathway downstream of the insulin receptor, DAF-2. Phospholipid products of AGE-1/PI3K activate AKT/PKB kinase signaling via PDK-1. AKT/PKB signaling antagonizes nuclear translocation of the DAF-16/FOXO transcription factor. Reduced AGE-1/PI3K signaling permits DAF-16 to direct dauer larval arrest and promote long lifespan in adult animals. In order to study the downstream effectors of AGE-1/PI3K signaling in C. elegans, we conducted a genetic screen for mutations that suppress the constitutive dauer arrest phenotype of age-1(mg109) animals. Results This report describes mutations recovered in a screen for suppressors of the constitutive dauer arrest (daf-C) phenotype of age-1(mg109). Two mutations corresponded to alleles of daf-16. Two mutations were gain-of-function alleles in the genes, akt-1 and pdk-1, encoding phosphoinositide-dependent serine/threonine kinases. A fifth mutation, mg227, located on chromosome X, did not correspond to any known dauer genes, suggesting that mg227 may represent a new component of the insulin pathway. Genetic epistasis analysis by RNAi showed that reproductive development in age-1(mg109);akt-1(mg247) animals was dependent on the presence of pdk-1. Similarly, reproductive development in age-1(mg109);pdk-1(mg261) animals was dependent on akt-1. However, reproductive development in age-1(mg109); mg227 animals required only akt-1, and pdk-1 activity was dispensable in this background. Interestingly, while mg227 suppressed dauer arrest in age-1(mg109) animals, it enhanced the long lifespan phenotype. In contrast, akt-1(mg247) and pdk-1(mg261) did not affect lifespan or stress resistance, while both daf-16 alleles fully suppressed these phenotypes. Conclusion A screen for suppressors of PI3K mutant phenotypes identified activating mutations in two known pathway components, providing insights into their regulation. In particular, the interdependence of akt-1 and pdk-1, even in activated forms, supports the existence of AGE-1-independent pathways for these phospholipid-dependent kinases. Phenotypic analysis of these alleles shows that the larval and adult outputs of AGE-1/PI3K are fully separable in these mutants. PMID:17020605

EphB4 promotes or suppresses Ras/MEK/ERK pathway in a context-dependent manner: Implications for EphB4 as a cancer target.

PubMed

Xiao, Zhan; Carrasco, Rosa; Kinneer, Krista; Sabol, Darrin; Jallal, Bahija; Coats, Steve; Tice, David A

2012-06-01

EphB4 is a member of the Eph receptor tyrosine kinase family shown to act in neuronal guidance and mediate venal/arterial separation. In contrast to these more established roles, EphB4's function in cancer is much less clear. Here we illustrate both tumor promoting as well as suppressing roles of EphB4, by showing that its activation resulted in inhibition of the Ras/ERK pathway in endothelial cells but activation of the same pathway in MCF-7 breast cancer cells. This was true if EphB4 was stimulated with EphrinB2, its natural ligand, or an agonistic monoclonal antibody for EphB4. Correspondingly, EphB4 activation stimulated MCF7 growth while inhibiting HUVEC cell proliferation. The reason for these dramatic differences is due to functional coupling of EphB4 to different downstream effectors. Reduction of p120 RasGAP in HUVEC cells attenuated the inhibitory effect of EphB4 activation on the ERK pathway, whereas knockdown of PP2A in MCF7 cells attenuated EphB4 activation of the ERK pathway. This represents the first time a functional coupling between Eph receptor and PP2A has been demonstrated leading to activation of an oncogenic pathway. Our study illustrates the caveats and potential challenges of targeting EphB4 for cancer therapy due to the conflicting effects on cancer cell and endothelial cell compartments.

Effect of resistance exercise under conditions of reduced blood insulin on AMPKα Ser485/491 inhibitory phosphorylation and AMPK pathway activation.

PubMed

Kido, Kohei; Yokokawa, Takumi; Ato, Satoru; Sato, Koji; Fujita, Satoshi

2017-08-01

Insulin stimulates skeletal muscle glucose uptake via activation of the protein kinase B/Akt (Akt) pathway. Recent studies suggest that insulin downregulates AMP-activated protein kinase (AMPK) activity via Ser485/491 phosphorylation of the AMPK α-subunit. Thus lower blood insulin concentrations may induce AMPK signal activation. Acute exercise is one method to stimulate AMPK activation; however, no study has examined the relationship between blood insulin levels and acute resistance exercise-induced AMPK pathway activation. Based on previous findings, we hypothesized that the acute resistance exercise-induced AMPK pathway activation would be augmented by disruptions in insulin secretion through a decrease in AMPKα Ser485/491 inhibitory phosphorylation. To test the hypothesis, 10-wk-old male Sprague-Dawley rats were administered the toxin streptozotocin (STZ; 55 mg/kg) to destroy the insulin secreting β-cells. Three days postinjection, the right gastrocnemius muscle from STZ and control rats was subjected to resistance exercise by percutaneous electrical stimulation. Animals were killed 0, 1, or 3 h later; activation of the Akt/AMPK and downstream pathways in the muscle tissue was analyzed by Western blotting and real-time PCR. Notably, STZ rats showed a significant decrease in basal Akt and AMPKα Ser485/491 phosphorylation, but substantial exercise-induced increases in both AMPKα Thr172 and acetyl-CoA carboxylase (ACC) Ser79 phosphorylation were observed. Although no significant impact on resistance exercise-induced Akt pathway activation or glucose uptake was found, resistance exercise-induced peroxisome proliferator-activated receptor (PPAR)-γ coactivator-1 α (PGC-1α) gene expression was augmented by STZ treatment. Collectively, these data suggest that circulating insulin levels may regulate acute resistance exercise-induced AMPK pathway activation and AMPK-dependent gene expression relating to basal AMPKα Ser485/491 phosphorylation. Copyright © 2017 the American Physiological Society.