The gut microbiome is composed of an enormous number of microorganisms, generally regarded as commensal bacteria. Resident gut bacteria are an important contributor to health and significant evidence suggests that the presence of healthy and diverse gut microbiota is important for normal cognitive and emotional processing. Here we measured the expression of monoamine neurotransmitter-related genes in the hippocampus of germ-free (GF) mice and specific-pathogen-free (SPF) mice to explore the effect of gut microbiota on hippocampal monoamine functioning. In total, 19 differential expressed genes (Htr7, Htr1f, Htr3b, Drd3, Ddc, Maob, Tdo2, Fos, Creb1, Akt1, Gsk3a, Pik3ca, Pla2g5, Cyp2d22, Grk6, Ephb1, Slc18a1, Nr4a1, Gdnf) that could discriminate between the two groups were identified. Interestingly, GF mice displayed anxiolytic-like behavior compared to SPF mice, which were not reversed by colonization with gut microbiota from SPF mice. Besides, colonization of adolescent GF mice by gut microbiota was not sufficient to reverse the altered gene expression associated with their GF status. Taking these findings together, the absence of commensal microbiota during early life markedly affects hippocampal monoamine gene-regulation, which was associated with anxiolytic behaviors and monoamine neurological signs.

OBJECTIVE

Selenoprotein T (SelT or SELENOT) is a novel thioredoxin-like enzyme whose genetic ablation in mice results in early embryonic lethality. SelT exerts an essential cytoprotective action during development and after injury through its redox-active catalytic site. This study aimed to determine the expression and regulation of SelT in the mammalian heart in normal and pathological conditions and to evaluate the cardioprotective effect of a SelT-derived peptide, SelT43-52(PSELT) encompassing the redox motif which is key to its function, against ischaemia/reperfusion(I/R) injury.

METHODS

We used the isolated Langendorff rat heart model and different analyses by immunohistochemistry, Western blot and ELISA.

RESULTS

We found that SelT expression is very abundant in embryo but is undetectable in adult heart. However, SelT expression was tremendously increased after I/R. PSELT (5 nmol/L) was able to induce pharmacological post-conditioning cardioprotection as evidenced by a significant recovery of contractility (dLVP) and reduction of infarct size (IS), without changes in cardiac contracture (LVEDP). In contrast, a control peptide lacking the redox site did not confer cardioprotection. Immunoblot analysis showed that PSELT-dependent cardioprotection is accompanied by a significant increase in phosphorylated Akt, Erk-1/2 and Gsk3α-β, and a decrement of p38MAPK. PSELT inhibited the pro-apoptotic factors Bax, caspase 3 and cytochrome c and stimulated the anti-apoptotic factor Bcl-2. Furthermore, PSELT significantly reduced several markers of I/R-induced oxidative and nitrosative stress.

CONCLUSIONS

These results unravel the role of SelT as a cardiac modulator and identify PSELT as an effective pharmacological post-conditioning agent able to protect the heart after ischaemic injury.

Bisphenol A (BPA), a member of the environmental endocrine disruptors (EDCs), has recently received increased attention because of its effects on brain insulin resistance. Available data have indicated that brain insulin resistance may contribute to neurodegenerative diseases. However, the associated mechanisms that underlie BPA-induced brain-related outcomes remain largely unknown. In the present study, we identified significant insulin signaling disturbances in the SH-SY5Y cell line that were mediated by BPA, including the inhibition of physiological p-IR Tyr1355 tyrosine, p-IRS1 tyrosine 896, p-AKT serine 473 and p-GSK3α/β serine 21/9 phosphorylation, as well as the enhancement of IRS1 Ser307 phosphorylation; these effects were clearly attenuated by insulin and rosiglitazone. Intriguingly, Alzheimer's disease (AD)-associated pathological proteins, such as BACE-1, APP, β-CTF, α-CTF, Aβ 1-42 and phosphorylated tau proteins (S199, S396, T205, S214 and S404), were substantially increased after BPA exposure, and these effects were abrogated by insulin and rosiglitazone treatment; these findings underscore the specific roles of insulin signaling in BPA-mediated AD-like neurotoxicity. Thus, an understanding of the regulation of insulin signaling may provide novel insights into potential therapeutic targets for BPA-mediated AD-like neurotoxicity.

Here we explored the antitumor-activity of novel-formulated-form of curcumin (phytosomal-encapsulated-curcumin) or in combination with 5-FU in breast cancer. The antiproliferative activity was assessed in 2D and 3-dimensional cell-culture-model. The migratory-behaviors of the cells were determined by migration assay. The expression levels of CyclinD1,GSK3a/b, P-AMPK, MMP9, and E-cadherin were studied by qRT-PCR and/or Western blotting. The anti-inflammatory of nano-curcumin was assessed, while antioxidant activity was evaluated by malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and total thiols (T-SH). To understand dynamic behavior of genes, we reconstructed a Boolean network, while the robustness of this model was evaluated by Hamming distance. phytosomal-curcumin suppressed cell-growth followed by tumor-shrinkage in 3D model through perturbation of AMP-activated protein kinase. Curcumin reduced the invasiveness of MCF-7 through perturbation of E-cadherin. Moreover, phytosomal-curcumin inhibited the tumor growth in xerograph model. Histological staining of tumor tissues revealed vascular disruption and RBC extravasation, necrosis, tumor stroma, and inflammation. Co-treatment of curcumin and 5-FU reduced the lipid-peroxidation and increased MDA/SOD level. Of note, curcumin reduced cyclinD-expression in breast cancer cell treated with thrombin, and activates AMPK in a time-dependent manner. Also suppression of AMPK abrogated inhibitory effect of phytosomal-curcumin on thrombin-induced cyclin D1 over-expression, suggesting that AMPK is essential for anti-proliferative effect of this agent in breast cancer. Our finding demonstrated that phytosomal-curcumin antagonizes cell growth and migration, induced by thrombin through AMP-Kinase in breast cancer, supporting further-investigations on the therapeutic potential of this novel anticancer agent in treatment of breast cancer.

It was reported that lumican inhibits the activity of metalloproteinase MMP-14 and melanoma cell migration in vitro and in vivo. Moreover, Snail triggers epithelial-to-mesenchymal transition and the metastatic potential of cancer cells. Therefore, the aim of this study was to examine the effect of lumican on Mock and Snail overexpressing melanoma B16F1 cells in vivo. Lung metastasis was analyzed after intravenous injections of Mock-B16F1 and Snail-B16F1 cells in Lum^+/+ and Lum^-/- mice. At day 14, mice were sacrificed, and lungs were collected. The number of lung metastatic nodules was significantly higher in mice injected with Snail-B16F1 cells as compared to mice injected with Mock-B16F1 cells confirming the pro-metastatic effect of Snail. This effect was stronger in Lum^-/- mice as compared to Lum^+/+, suggesting that endogenous lumican of wild-type mice significantly inhibits metastasis to lungs. Scanning electron and confocal microscopy investigations demonstrated that lumican inhibits the development of elongated cancer cell phenotypes which are known to develop invadopodia releasing MMPs. Moreover, lumican was shown to affect the expression of cyclin D1, cortactin, vinculin, hyaluronan synthase 2, heparanase, MMP-14 and the phosphorylation of FAK, AKT, p130 Cas and GSK3α/β. Altogether, these data demonstrated that lumican significantly inhibits lung metastasis in vivo, as well as cell invasion in vitro, suggesting that a lumican-based strategy targeting Snail-induced metastasis could be useful for melanoma treatment.

Keywords: epithelial-to-mesenchymal transition; invadopodia; lumican; lung metastasis; melanoma; snail.

We have developed an optimized protocol for plasma targeted mRNA sequencing in our previous study. Here, we performed plasma targeted mRNA sequencing for 40 colorectal adenoma patients and 39 colonoscopy-proven normal controls in order to find potential circulating mRNA markers for colorectal adenoma. Results showed that GSK3A and RHOA were differential expressed genes identified by a cut-off of fold change >2 and adjusted P value < 0.05. More detailed analysis showed that the expression of both GSK3A (0.01-fold with adjusted P < 1 × 10^-6) and RHOA (0.35-fold with adjusted P < 0.01) in adenoma patients was significantly lower than those in normal healthy subjects. Based on the enrichment analysis of biological process for potential markers, we found that the regulation of programmed cell death (GO: 0043067; GO: 0043069), regulation of cell death (GO: 0010941; GO: 0060548) and cell differentiation (GO: 0021861) were the main processes involved in adenoma formation. In summary, this study is a cutting-edge research on the detection of plasma mRNA in colorectal adenoma patients and normal healthy subjects.

Although memories appear to be elusive phenomena, they are stored in the network of physical connections between neurons. Dendritic spines, which are actin-rich dendritic protrusions, serve as the contact points between networked neurons. The spines' shape contributes to the strength of signal transmission. To acquire and store information, dendritic spines must remain plastic, i.e., able to respond to signals, by changing their shape. We asked whether glycogen synthase kinase (GSK) 3α and GSK3β, which are implicated in diseases with neuropsychiatric symptoms, such as Alzheimer's disease, bipolar disease and schizophrenia, play a role in a spine structural plasticity. We used Latrunculin B, an actin polymerization inhibitor, and chemically induced Long-Term Depression to trigger fast spine shape remodeling in cultured hippocampal neurons. Spine shrinkage induced by either stimulus required GSK3α activity. GSK3β activity was only important for spine structural changes after treatment with Latrunculin B. Our results indicate that GSK3α is an essential component for short-term spine structural plasticity. This specific function should be considered in future studies of neurodegenerative diseases and neuropsychiatric conditions that originate from suboptimal levels of GSK3α/β activity.

Physical exercise induces inflammatory and oxidative markers production in the skeletal muscle and this process is under the control of both endogenous and exogenous modulators. Recently, molecular hydrogen (H2) has been described as a therapeutic gas able to reduced oxidative stress in a number of conditions. However, nothing is known about its putative role in the inflammatory and oxidative status during a session of acute physical exercise in sedentary rats. Therefore, we tested the hypothesis that H2 attenuates both inflammation and oxidative stress induced by acute physical exercise. Rats ran at 80% of their maximum running velocity on a closed treadmill inhaling either the H2 gas (2% H2, 21% O2, balanced with N2) or the control gas (0% H2, 21% O2, balanced with N2) and were euthanized immediately or 3 h after exercise. We assessed plasma levels of inflammatory cytokines [tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6] and oxidative markers [superoxide dismutase (SOD), thiobarbituric acid reactive species (TBARS) and nitrite/nitrate (NOx)]. In addition, we evaluated the phosphorylation status of intracellular signaling proteins [glycogen synthase kinase type 3 (GSK3α/β) and the cAMP responsive element binding protein (CREB)] that modulate several processes in the skeletal muscle during exercise, including changes in exercise-induced reactive oxygen species (ROS) production. As expected, physical exercise increased virtually all the analyzed parameters. In the running rats, H2 blunted exercise-induced plasma inflammatory cytokines (TNF-α and IL-6) surges. Regarding the oxidative stress markers, H2 caused further increases in exercise-induced SOD activity and attenuated the exercise-induced increases in TBARS 3 h after exercise. Moreover, GSK3α/β phosphorylation was not affected by exercise or H2 inhalation. Otherwise, exercise caused an increased CREB phosphorylation which was attenuated by H2. These data are consistent with the notion that H2 plays a key role in decreasing exercise-induced inflammation, oxidative stress, and cellular stress.

BACKGROUND

The placenta is metabolically highly active due to extensive endocrine and active transport functions. Hence, placental tissues soon become ischaemic after separation from the maternal blood supply. Ischaemia rapidly depletes intracellular ATP, and leads to activation of stress-response pathways aimed at reducing metabolic demands and conserving energy resources for vital functions. Therefore, this study aimed to elucidate the effects of ischaemia ex vivo as may occur during tissue collection on phosphorylation of placental proteins and kinases involved in growth and cell survival, and on mitochondrial complexes.

METHODS

Eight term placentas obtained from normotensive non-laboured elective caesarean sections were kept at room-temperature and sampled at 10, 20, 30 and 45 min after delivery. Samples were analyzed by Western blotting.

RESULTS

Between 10 and 45 min the survival signalling pathway intermediates, P-AKT, P-GSK3α and β, P-4E-BP1 and P-p70S6K were reduced by 30-65%. Stress signalling intermediates, P-eIF2α increased almost 3 fold after 45 min. However, other endoplasmic reticulum stress markers and the Heat Shock Proteins, HSP27, HSP70 and HSP90, did not change. Phosphorylation of AMPK, an energy sensor, was elevated 2 fold after 45 min. Contemporaneously, there was an ∼25% reduction in mitochondrial complex IV subunit I.

CONCLUSIONS

These results suggest that for placental signalling studies, samples should be taken and processed within 10 min of caesarean delivery to minimize the impact of ischaemia on protein phosphorylation.

Preterm birth remains the largest single cause of neonatal death and morbidity. Infection and/or inflammation are strongly associated with preterm delivery. Glycogen synthase kinase 3 (GSK3) is known to be a crucial mediator of inflammation homeostasis. The aims of this study were to determine the effect of spontaneous human labour in foetal membranes and myometrium on GSK3α/β expression, and the effect of inhibition of GSK3α/β on pro-labour mediators in foetal membranes and myometrium stimulated with Toll-like receptor (TLR) ligands and pro-inflammatory cytokines. Term and preterm labour in foetal membranes was associated with significantly decreased serine phosphorylated GSK3α and β expression, and thus increased GSK3 activity. There was no effect of term labour on serine phosphorylated GSK3β expression in myometrium. The specific GSK3α/β inhibitor CHIR99021 significantly decreased lipopolysaccharide (ligand to TLR4)-stimulated pro-inflammatory cytokine gene expression and release; COX2 gene expression and prostaglandin release; and MMP9 gene expression and pro MMP9 release in foetal membranes and/or myometrium. CHIR99021 also decreased FSL1 (TLR2 ligand) and flagellin (TLR5 ligand)-induced pro-inflammatory cytokine gene expression and release and COX2 mRNA expression and prostaglandin release. GSK3β siRNA knockdown in primary myometrial cells was associated with a significant decrease in IL1β and TNFα-induced pro-inflammatory cytokine and prostaglandin release. In conclusion, GSK3α/β activity is increased in foetal membranes after term and preterm labour. Pharmacological blockade of the kinase GSK3 markedly reduced pro-inflammatory and pro-labour mediators in human foetal membranes and myometrium, providing a possible therapeutics for the management of preterm labour.

Biliverdin reductase A (BVR) and Akt isozymes have overlapping pleiotropic functions in the insulin/PI3K/MAPK pathway. Human BVR (hBVR) also reduces the hemeoxygenase activity product biliverdin to bilirubin and is directly activated by insulin receptor kinase (IRK). Akt isoenzymes (Akt1-3) are downstream of IRK and are activated by phosphatidylinositol-dependent kinase 1 (PDK1) phosphorylating T(308) before S(473) autophosphorylation. Akt (RxRxxSF) and PDK1 (RFxFPxFS) binding motifs are present in hBVR. Phosphorylation of glycogen synthase kinase 3 (GSK3) isoforms α/β by Akts inhibits their activity; nonphosphorylated GSK3β inhibits activation of various genes. We examined the role of hBVR in PDK1/Akt1/GSK3 signaling and Akt1 in hBVR phosphorylation. hBVR activates phosphorylation of Akt1 at S(473) independent of hBVR's kinase competency. hBVR and Akt1 coimmunoprecipitated, and in-cell Förster resonance energy transfer (FRET) and glutathione S-transferase pulldown analyses identified Akt1 pleckstrin homology domain as the interactive domain. hBVR activates phosphorylation of Akt1 at S(473) independent of hBVR's kinase competency. Site-directed mutagenesis, mass spectrometry, and kinetic analyses identified S(230) in hBVR (225)RNRYLSF sequence as the Akt1 target. Underlined amino acids are the essential residues of the signaling motifs. In cells, hBVR-activated Akt1 increased both GSK3α/β and forkhead box of the O class transcription class 3 (FoxO3) phosphorylation and inhibited total GSK3 activity; depletion of hBVR released inhibition and stimulated glucose uptake. Immunoprecipitation analysis showed that PDK1 and hBVR interact through hBVR's PDK1 binding (161)RFGFPAFS motif and formation of the PDK1/hBVR/Akt1 complex. sihBVR blocked complex formation. Findings identify hBVR as a previously unknown coactivator of Akt1 and as a key mediator of Akt1/GSK3 pathway, as well as define a key role for hBVR in Akt1 activation by PDK1.-Miralem, T., Lerner-Marmarosh, N., Gibbs, P. E. M., Jenkins, J. L., Heimiller, C., Maines, M. D. Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation.

The microtubule-associated protein Tau is responsible for a large group of neurodegenerative disorders, known as tauopathies, including Alzheimer's disease. Tauopathy result from augmented and/or aberrant phosphorylation of Tau. Besides aging and various genetic and epigenetic defects that remain largely unknown, an important non-genetic agent that contributes is hypothermia, eventually caused by anesthesia. Remarkably, tauopathy in brains of hibernating mammals is not pathogenic, and, because it is fully reversible, is even considered to be neuroprotective. Here, we assessed the terminal phase of Tau.P301L mice and bigenic crosses with mice lacking glycogen synthase kinase 3 (GSK3)α completely, or GSK3β specifically in neurons. We also analysed biGT bigenic mice that co-express Tau.P301L with GSK3β.S9A and develop severe forebrain tauopathy with age. We found that the precocious mortality of Tau.P301L mice was typified by hypothermia that aggravated Tau phosphorylation, but, surprisingly, independently of GSK3α/β. The important contribution of hypothermia at the time of death of mice with tauopathy suggests that body temperature should be included as a parameter in the analysis of pre-clinical models, and, by extension, in patients suffering from tauopathy.

GSK3β binding of GSKIP affects neurite outgrowth, but the physiological significance of PKA binding to GSKIP remains to be determined. We hypothesized that GSKIP and GSK3β mediate cAMP/PKA/Drp1 axis signaling and modulate mitochondrial morphology by forming a working complex comprising PKA/GSKIP/GSK3β/Drp1. We demonstrated that GSKIP wild-type overexpression increased phosphorylation of Drp1 S637 by 7-8-fold compared to PKA kinase-inactive mutants (V41/L45) and a GSK3β binding-defective mutant (L130) under H2O2 and forskolin challenge in HEK293 cells, indicating that not only V41/L45, but also L130 may be involved in Drp1-associated protection of GSKIP. Interestingly, silencing either GSKIP or GSK3β but not GSK3α resulted in a dramatic decrease in Drp1 S637 phosphorylation, revealing that both GSKIP and GSK3β are required in this novel PKA/GSKIP/GSK3β/Drp1 complex. Moreover, overexpressed kinase-dead GSK3β-K85R, which retains the capacity to bind GSKIP, but not K85M which shows total loss of GSKIP-binding, has a higher Drp1 S637 phosphorylation similar to the GSKIP wt overexpression group, indicating that GSK3β recruits Drp1 by anchoring rather than in a kinase role. With further overexpression of either V41/L45P or the L130P GSKIP mutant, the elongated mitochondrial phenotype was lost; however, ectopically expressed Drp1 S637D, a phosphomimetic mutant, but not S637A, a non-phosphorylated mutant, restored the elongated mitochondrial morphology, indicating that Drp1 is a downstream effector of direct PKA signaling and possibly has an indirect GSKIP function involved in the cAMP/PKA/Drp1 signaling axis. Collectively, our data revealed that both GSKIP and GSK3β function as anchoring proteins in the cAMP/PKA/Drp1 signaling axis modulating Drp1 phosphorylation.

We tested the hypothesis that glycogen synthase kinase 3α/β (GSK3α/β) modulates tumor necrosis factor-a (TNF) induced increased lung vascular permeability. Rats were treated with TNF (i.v., ~100ng/ml) or vehicle 0.5h, 4.0h and 24.0h prior to lung isolation. Rats were co-treated with the GSK3α/β inhibitors SB216763 (0.6mg/kg) or TDZD-8 (1.0mg/kg). After TNF, the isolated lung was assessed for hemodynamics, wet-dry/dry weight (W-D/D) and extravascular albumin. Extravascular albumin significantly increased at TNF-24h compared to Control. In the GSK3α/β-inhibited+TNF groups, extravascular albumin was similar to the Control and respective SB216763 and TDZD-8 groups. In separate studies, to assess GSK3α/β-activity, lung lysate was assessed for phospho-GSK3α/β-Ser(21/9), total GSK3α/β, un-phospho-β-catenin-Ser(33/37) and total β-catenin. In the TNF-4.0h group, there was no change in GSK3α/phospho-GSK3α-Ser(21) but there was an increase in GSK3β/GSK3β-Ser(9) compared to Control, indicating GSK3β activation at TNF-4.0h. GSK3β activation was verified because there was a decrease in un-phospho-β-catenin-Ser(33/37)/β-catenin in the TNF-4.0 group, a specific outcome for GSK3β activation. In the SB216763+TNF group, un-phospho-β-catenin-Ser(33/37) was similar to Control, indicating prevention of TNF-induced GSK3β activation. In the TNF-24h group, there were increases in the biomarkers of inflammation phospho-eNOS-Ser (1117) and oxidized protein, which did not occur in the SB216763+TNF-24h and TDZD-8+TNF-24h groups. In the SB216763+TNF-24h and TDZD-8+TNF-24h groups, un-phospho-β-catenin-Ser(33/37) was greater than in the Control, indicating continued inhibition of GSK3β. The data indicates that pharmacologic inhibition of GSK3β inhibits TNF induced increased endothelial permeability associated with lung inflammation.

The function of dendritic cells (DCs) is modified by glycogen synthase kinase GSK3 and GSK3 inhibitors have been shown to protect against inflammatory disease. Regulators of GSK3 include the phosphoinositide 3 kinase (PI3K) pathway leading to activation of protein kinase B (PKB/Akt) and serum and glucocorticoid inducible kinase (SGK) isoforms, which in turn phosphorylate and thus inhibit GSK3. The present study explored, whether PKB/SGK-dependent inhibition of GSK3 contributes to the regulation of cytosolic Ca(2+) concentration following stimulation with bacterial lipopolysaccharides (LPS). To this end DCs from mutant mice, in which PKB/SGK-dependent GSK3α,β regulation was disrupted by replacement of the serine residues in the respective SGK/PKB-phosphorylation consensus sequence by alanine (gsk3(KI)), were compared to DCs from respective wild type mice (gsk3(WT)). According to Western blotting, GSK3 phosphorylation was indeed absent in gsk3(KI) DCs. According to flow cytometry, expression of antigen-presenting molecule major histocompatibility complex II (MHCII) and costimulatory molecule CD86, was similar in unstimulated and LPS (1μg/ml, 24h)-stimulated gsk3(WT) and gsk3(KI) DCs. Moreover, production of cytokines IL-6, IL-10, IL-12 and TNFα was not significantly different in gsk3(KI) and gsk3(WT) DCs. In gsk3(WT) DCs, stimulation with LPS (1μg/ml) within 10min led to transient phosphorylation of GSK3. According to Fura2 fluorescence, LPS (1μg/ml) increased cytosolic Ca(2+) concentration, an effect significantly more pronounced in gsk3(KI) DCs than in gsk3(WT) DCs. Conversely, GSK3 inhibitor SB216763 (3-[2,4-Dichlorophenyl]-4-[1-methyl-1H-indol-3-yl]-1H-pyrrole-2,5-dione, 10μM, 30min) significantly blunted the increase of cytosolic Ca(2+) concentration following LPS exposure. In conclusion, PKB/SGK-dependent GSK3α,β activity participates in the regulation of Ca(2+) signaling in dendritic cells.

PCI-24781 is a novel histone deacetylase inhibitor that inhibits tumor proliferation and promotes cell apoptosis. However, it is unclear whether PCI-24781 inhibits Enhancer of Zeste 2 (EZH2) expression in malignant gliomas. In this work, three glioma cell lines were incubated with various concentrations of PCI-24781 (0, 0.25, 0.5, 1, 2.5 and 5 μM) and analyzed for cell proliferation by the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay and colony formation, and cell cycle and apoptosis were assessed by flow cytometry. The expression of EZH2 and apoptosis-related proteins was assessed by western blotting. Malignant glioma cells were also transfected with EZH2 siRNA to examine how PCI-24781 suppresses tumor cells. EZH2 was highly expressed in the three glioma cell lines. Incubation with PCI-24781 reduced cell proliferation and increased cell apoptosis by down-regulating EZH2 in a concentration-dependent manner. These effects were simulated by EZH2 siRNA. In addition, PCI-24781 or EZH2 siRNA accelerated cell apoptosis by down-regulating the expression of AKT, mTOR, p70 ribosomal protein S6 kinase (p70s6k), glycogen synthase kinase 3A and B (GSK3a/b) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1). These data suggest that PCI-24781 may be a promising therapeutic agent for treating gliomas by down-regulating EZH2 which promotes cell apoptosis by suppressing the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of the rapamycin (mTOR) pathway.

The bacterial protein tyrosine phosphatase PtpA is a key virulence factor released by Mycobacterium tuberculosis in the cytosol of infected macrophages. So far only two unrelated macrophage components (VPS33B, GSK3α) have been identified as PtpA substrates. As tyrosine phosphatases are capable of using multiple substrates, we developed an improved methodology to pull down novel PtpA substrates from an enriched P-Y macrophage extract using the mutant PtpA D126A. This methodology reduced non-specific protein interactions allowing the identification of four novel putative PtpA substrates by MALDI-TOF-MS and nano LC-MS: three mitochondrial proteins - the trifunctional enzyme (TFP), the ATP synthase, and the sulfide quinone oxidoreductase - and the cytosolic 6-phosphofructokinase. All these proteins play a relevant role in cell energy metabolism. Using surface plasmon resonance, PtpA was found to bind immunopurified human TFP through its catalytic site since TFP-PtpA association was inhibited by a specific phosphatase inhibitor. Moreover, PtpA wt was capable of dephosphorylating immunopurified human TFP in vitro supporting that TFP may be a bona fide PtpA susbtrate. Overall, these results suggest a novel scenario where PtpA-mediated dephosphorylation may affect pathways involved in cell energy metabolism, particularly the beta oxidation of fatty acids through modulation of TFP activity and/or cell distribution.

A series of chemically modified 7-phenylpyrrolo[3,2-f]quinolinones was synthesized and evaluated as anticancer agents. Among them, the most cytotoxic (subnanomolar GI50 values) amidic derivative 5f was shown to act as an inhibitor of tubulin polymerization (IC50, 0.99 μM) by binding to the colchicine site with high affinity. Moreover, 5f induced cell cycle arrest in the G2/M phase of the cell cycle in a concentration dependent manner, followed by caspase-dependent apoptotic cell death. Compound 5f also showed lower toxicity in nontumoral cells, suggesting selectivity toward cancer cells. Additional experiments revealed that 5f inhibited the enzymatic activity of multiple kinases, including AURKA, FLT3, GSK3A, MAP3K, MEK, RSK2, RSK4, PLK4, ULK1, and JAK1. Computational studies showed that 5f can be properly accommodated in the colchicine binding site of tubulin as well as in the ATP binding clefts of all examined kinases. Our data indicate that the excellent antiproliferative profile of 5f may be derived from its interactions with multiple cellular targets.

OBJECTIVE

Aberrant c-Myc activity plays a central role in cancer transformation. γ-tocotrienol is a member of the vitamin E family that displays potent anti-cancer activity. Here, studies were conducted to determine the role of c-Myc in mediating anti-proliferative effects of γ-tocotrienol in mammary cancer cells.

METHODS

Treatment effects on mouse +SA and human MCF-7 mammary cancer cell proliferation were determined by MTT assay and Ki-67 staining. Protein expression was determined by western blot analysis. Immunofluorescence staining and qRT-PCR were used to characterize cellular c-Myc and MYC levels respectively.

RESULTS

Anti-proliferative effects of γ-tocotrienol were associated with reduction in total c-Myc and phosphorylated-c-Myc-serine 62, and increase in phosphorylated-c-Myc-threonine 58 levels. γ-tocotrienol also reduced PI3K/Akt/mTOR and Ras/MEK/Erk mitogenic signalling, cyclin D1 and cyclin-dependent kinase 4 levels, and increased p27 levels. However, γ-tocotrienol had no effect on MYC mRNA levels. γ-tocotrienol also increased levels of FBW7 (E3 ligase that initiates ubiquitination of c-Myc), but had no effect on serine/threonine phosphatase PP2A or isomerase Pin 1 levels. Combined treatment with GSK3α/β inhibitor LiCl or proteasome inhibitor MG132 blocked γ-tocotrienol-induced reductions in c-Myc.

CONCLUSIONS

These findings indicate that anti-proliferative effects of γ-tocotrienol are associated with reduction in c-Myc that results from increase in GSK-3α/β-dependent ubiquitination and degradation, rather than from reduction in c-Myc synthesis in +SA and MCF-7 mammary cancer cells.

Glycogen synthase kinase 3 (GSK3) β and α are serine/threonine kinases involved in many biological processes. A primary mechanism of GSK3 activity regulation is phosphorylation of N-terminal serine (S) residues (S9 in GSK3β, S21 in GSK3α). Phosphorylation is inhibitory to GSK3 kinase activity because the phosphorylated N-terminus acts as a competitive inhibitor for primed substrates. Despite widespread interest in GSK3 across most fields of biology, the research community does not have reagents that specifically react with nonphosphoS9/21 GSK3β/α (the so-called "active" form). Here, we describe two novel monoclonal antibodies that specifically react with nonphosphoS9/21 GSK3β/α in multiple species (human, mouse, and rat). One of the antibodies is specific for nonphospho-S9 GSK3β (clone 12B2) and one for nonphospho-S9/21 GSK3β/α (clone 15C2). These reagents were validated for specificity and reactivity in several biochemical and immunochemical assays, and they show linear detection of nonphosphoS GSK3. Finally, these reagents provide significant advantages in studying GSK3β regulation. We used both antibodies to study the regulation of S9 phosphorylation by Akt and protein phosphatases. We used 12B2 (due to its specificity for GSK3β) and to demonstrate that protein phosphatase inhibition reduces nonphospho-S9 GSK3β levels and lowers kinase activity within cells. The ability to use the same reagent across biochemical, immunohistological and kinase activity assays provides a powerful approach for studying serine-dependent regulation of GSK3β/α.

Downstream of growth factor receptors, signaling by the phosphoinositide 3 kinase (PI3K) pathway is known to play an important role in the growth and survival of many tumor types. The PI3K pathway simplistically comprises PI3K itself, followed by PDK-1, then AKT and finally glycogen synthase kinase 3 (GSK3). PI3K/AKT signaling promotes increased GSK3 phosphorylation, that is associated with reduced GSK3 activity. There are two isoforms of GSK3, GSK3α and GSK3β, which have a high degree of sequence homology. GSK3 plays a role not only in the regulation of glycogen synthase activity but in the expression of multiple other proteins that play a role in cancer biology, including cyclins and anti-apoptotic proteins.

Melanoma is one of the most highly mutated cancer types. To identify functional drivers of melanoma, we searched for cross-species conserved mutations utilizing a mouse melanoma model driven by loss of PTEN and CDKN2A, and identified mutations in Kras, Erbb3, and Ptpn11. PTPN11 encodes the SHP2 protein tyrosine phosphatase (PTP) that activates the RAS/RAF/MAPK pathway. Although PTPN11 is an oncogene in leukemia, lung, and breast cancers, its roles in melanoma are not clear. In this study, we found that PTPN11 is frequently activated in human melanoma specimens and cell lines and is required for full RAS/RAF/MAPK signaling activation in BRAF wild-type (either NRAS mutant or wild-type) melanoma cells. PTPN11 played oncogenic roles in melanoma by driving anchorage-independent colony formation and tumor growth. In Pten and Cdkn2a null mice, tet-inducible and melanocyte-specific PTPN11 E76K expression significantly enhanced melanoma tumorigenesis. Melanoma cells derived from this mouse model showed doxycycline-dependent tumor growth in nude mice. Silencing PTPN11 E76K expression by doxycycline withdrawal caused regression of established tumors by induction of apoptosis and senescence, and suppression of proliferation. Moreover, the PTPN11 inhibitor (SHP099) also caused regression of NRAS Q61K-mutant melanoma. Using a quantitative tyrosine phosphoproteomics approach, we identified GSK3α/β as one of the key substrates that were differentially tyrosine-phosphorylated in these experiments modulating PTPN11. This study demonstrates that PTPN11 plays oncogenic roles in melanoma and regulates RAS and GSK3β signaling pathways. Implications: This study identifies PTPN11 as an oncogenic driver and a novel and actionable therapeutic target for BRAF wild-type melanoma.

OBJECTIVE

Restoration of coronary blood flow is crucial in the treatment of acute myocardial infarction. Reperfusion, however, induces ischaemia-reperfusion (IR) injury, which further deteriorates myocardial function. The innate immune system plays an important role in this process, mediating rapid influx of immune cells into the reperfused myocardium. Leukotriene B4 is an important leucocyte chemoattractant, performing its actions through binding to its specific receptor BLT1. We hypothesized that treatment with LSN2792613, a selective BLT1 antagonist, reduces infarct size (IS) in a mouse model of myocardial IR injury.

RESULTS

Male C57Bl/6J mice were subjected to myocardial ischaemia for 30 min by surgical coronary artery ligation, followed by reperfusion. Mice received either LSN2792613 or vehicle, three times daily (orally) for up to 72 h after reperfusion. BLT1 inhibition with LSN2792613 reduced IS compared with vehicle treatment (26.9 ± 2.7 vs. 34.9 ± 2.2%, P = 0.030) at 24 h after reperfusion. The levels of IL-6 and keratinocyte chemoattractant were reduced in the infarcted tissue of LSN2792613-treated mice. Reduced apoptosis in LSN2792613-treated mice was suggested by increased levels of phosphorylated JNK and GSK3α/β, and confirmed by flow cytometric analysis showing less apoptotic and necrotic cardiomyocytes in the infarcted myocardium. Echocardiography at 4 weeks after myocardial IR showed a slightly higher ejection fraction and stroke volume in mice treated with LSN2792613 compared with vehicle-treated mice, whereas left ventricular volumes were comparable.

CONCLUSIONS

Selective BLT1 inhibition with LSN2792613 reduces inflammation and apoptosis following IR, resulting in reduced IS, and therefore might be a promising strategy to prevent myocardial IR injury.

BACKGROUND

Haemonchus contortus is a common bloodsucking nematode causing widespread economic loss in agriculture. Upon H. contortus infection, a series of host responses is elicited, especially those related to T lymphocyte immunity. Existing studies mainly focus on the general immune responses of sheep T lymphocyte to H. contortus, lacking investigations at the molecular level. The objective of this study was to obtain a systematic transcriptional profiling of the T lymphocytes in H. contortus primary-infected sheep.

METHODS

Nematode-free sheep were orally infected once with H. contortus L3s. T lymphocyte samples were collected from the peripheral blood of 0, 3, 30 and 60 days post infection (dpi) infected sheep. Microarrays were used to compare gene transcription levels between samples. Quantitative RT-PCR was employed to validate the microarray data. Gene Ontology and KEGG pathway analysis were utilized for the annotation of differentially expressed genes.

RESULTS

Our microarray data was consistent with qPCR results. From microarrays, 853, 242 and 42 differentially expressed genes were obtained in the 3d vs. 0d, 30d vs. 0d and 60d vs. 0d comparison groups, respectively. Gene Ontology and KEGG pathway analysis indicated that these genes were involved in metabolism, signaling, cell growth and immune system processes. Functional analysis of significant differentially expressed genes, such as SLC9A3R2, ABCB9, COMMD4, SUGT1, FCER1G, GSK3A, PAK4 and FCER2, revealed a crucial association with cellular homeostasis maintenance and immune response. Our data suggested that maintaining both effective immunological response and natural cellular activity are important for T lymphocytes in fighting against H. contortus infection.

CONCLUSIONS

Our results provide a substantial list of candidate genes in sheep T lymphocytes response to H. contortus infection, and contribute novel insights into a general immune response upon infection.