ResearchPad - signal-inhibition https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[The transition from local to global patterns governs the differentiation of mouse blastocysts]]> https://www.researchpad.co/article/elastic_article_14745 During mammalian blastocyst development, inner cell mass (ICM) cells differentiate into epiblast (Epi) or primitive endoderm (PrE). These two fates are characterized by the expression of the transcription factors NANOG and GATA6, respectively. Here, we investigate the spatio-temporal distribution of NANOG and GATA6 expressing cells in the ICM of the mouse blastocysts with quantitative three-dimensional single cell-based neighbourhood analyses. We define the cell neighbourhood by local features, which include the expression levels of both fate markers expressed in each cell and its neighbours, and the number of neighbouring cells. We further include the position of a cell relative to the centre of the ICM as a global positional feature. Our analyses reveal a local three-dimensional pattern that is already present in early blastocysts: 1) Cells expressing the highest NANOG levels are surrounded by approximately nine neighbours, while 2) cells expressing GATA6 cluster according to their GATA6 levels. This local pattern evolves into a global pattern in the ICM that starts to emerge in mid blastocysts. We show that FGF/MAPK signalling is involved in the three-dimensional distribution of the cells and, using a mutant background, we further show that the GATA6 neighbourhood is regulated by NANOG. Our quantitative study suggests that the three-dimensional cell neighbourhood plays a role in Epi and PrE precursor specification. Our results highlight the importance of analysing the three-dimensional cell neighbourhood while investigating cell fate decisions during early mouse embryonic development.

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<![CDATA[Role of tumor-associated neutrophils in regulation of tumor growth in lung cancer development: A mathematical model]]> https://www.researchpad.co/article/5c58d634d5eed0c4840318a9

Neutrophils display rapid and potent innate immune responses in various diseases. Tumor-associated neutrophils (TANs) however either induce or overcome immunosuppressive functions of the tumor microenvironment through complex tumor-stroma crosstalk. We developed a mathematical model to address the question of how phenotypic alterations between tumor suppressive N1 TANS, and tumor promoting N2 TANs affect nonlinear tumor growth in a complex tumor microenvironment. The model provides a visual display of the complex behavior of populations of TANs and tumors in response to various TGF-β and IFN-β stimuli. In addition, the effect of anti-tumor drug administration is incorporated in the model in an effort to achieve optimal anti-tumor efficacy. The simulation results from the mathematical model were in good agreement with experimental data. We found that the N2-to-N1 ratio (N21R) index is positively correlated with aggressive tumor growth, suggesting that this may be a good prognostic factor. We also found that the antitumor efficacy increases when the relative ratio (Dap) of delayed apoptotic cell death of N1 and N2 TANs is either very small or relatively large, providing a basis for therapeutically targeting prometastatic N2 TANs.

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<![CDATA[Individual response to mTOR inhibition in delaying replicative senescence of mesenchymal stromal cells]]> https://www.researchpad.co/article/5c5ca2eed5eed0c48441edbc

Background aims

Delaying replicative senescence and extending lifespan of human mesenchymal stromal cells (MSCs) may enhance their potential for tissue engineering and cell based therapies. Accumulating evidence suggests that inhibitors of the mTOR signaling pathway, such as rapamycin, constitute promising pharmacological agents to retard senescence and extend stemness properties of various progenitor cell types. Here, we investigated whether the ability of rapamycin to postpone replicative senescence varies among bone marrow MSC samples (BM-MSCs) derived from different healthy donors, and explored the molecular mechanisms that drive rapamycin-mediated lifespan increment.

Methods

BM-MSCs at early passages were serially passaged either in absence or continuous presence of rapamycin and the number of cell population doublings until growth arrest was measured. The inhibition of mTOR signaling was assessed by the phosphorylation status of the downstream target RPS6. The expression levels of several senescence and pluripotency markers at early and late/senescent passages were analyzed by RT-qPCR, flow cytometry and western blot.

Results

We found that the lifespan extension in response to the continuous rapamycin treatment was highly variable among samples, but effective in most BM-MSCs. Despite all rapamycin-treated cells secreted significantly reduced levels of IL6, a major SASP cytokine, and expressed significantly higher levels of the pluripotency marker NANOG, the expression patterns of these markers were not correlated with the rapamycin-mediated increase in lifespan. Interestingly, rapamycin-mediated life-span extension was significantly associated only with repression of p16INK4A protein accumulation.

Conclusions

Taken together, our results suggest that some, but not all, BM-MSC samples would benefit from using rapamycin to postpone replicative arrest and reinforce a critical role of p16INK4A protein downregulation in this process.

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<![CDATA[Dissecting the roles of β-arrestin2 and GSK-3 signaling in 5-HT1BR-mediated perseverative behavior and prepulse inhibition deficits in mice]]> https://www.researchpad.co/article/5c633954d5eed0c484ae64c0

Serotonin-1B receptors (5-HT1BRs) modulate perseverative behaviors and prepulse inhibition (PPI) in humans and mice. These inhibitory G-protein-coupled receptors signal through a canonical G-protein-coupled pathway that is modulated by GSK-3β, and a noncanonical pathway mediated by the adaptor protein β-arrestin2 (Arrb2). Given the development of biased ligands that differentially affect canonical versus noncanonical signaling, we examined which signaling pathway mediates 5-HT1BR agonist-induced locomotor perseveration and PPI deficits, behavioral phenotypes observed in both obsessive-compulsive disorder (OCD) and autism spectrum disorder (ASD). To assess the role of canonical 5-HT1BR signaling, mice received acute pretreatment with a GSK-3 inhibitor (SB216763 or AR-A014418) and acute treatment with the 5-HT1A/1B receptor agonist RU24969 prior to assessing perseverative locomotor behavior in the open field, and PPI. To determine the role of noncanonical 5-HT1BR signaling, Arrb2 wild-type (WT), heterozygous (HT), and knockout (KO) mice received acute RU24969 treatment prior to behavioral testing. GSK-3 inhibition increased locomotor perseveration overall, and also failed to influence the RU24969-induced perseverative locomotor pattern in the open field. Yet, GSK-3 inhibition modestly reduced RU24969-induced PPI deficits. On the other hand, Arrb2 HT and KO mice showed reduced locomotion and no changes in perseveration overall, in addition to modest reductions in RU24969-induced locomotion and PPI deficits. In conclusion, our data do not support use of either GSK-3 inhibitors or β-arrestin2 inhibition in treatment of perseverative behaviors.

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<![CDATA[Ligand-activated PPARδ inhibits angiotensin II-stimulated hypertrophy of vascular smooth muscle cells by targeting ROS]]> https://www.researchpad.co/article/5c3e505ed5eed0c484d80f2b

We investigated the effect of peroxisome proliferator-activated receptor δ (PPARδ) on angiotensin II (Ang II)-triggered hypertrophy of vascular smooth muscle cells (VSMCs). Activation of PPARδ by GW501516, a specific ligand of PPARδ, significantly inhibited Ang II-stimulated protein synthesis in a concentration-dependent manner, as determined by [3H]-leucine incorporation. GW501516-activated PPARδ also suppressed Ang II-induced generation of reactive oxygen species (ROS) in VSMCs. Transfection of small interfering RNA (siRNA) against PPARδ significantly reversed the effects of GW501516 on [3H]-leucine incorporation and ROS generation, indicating that PPARδ is involved in these effects. By contrast, these GW501516-mediated actions were potentiated in VSMCs transfected with siRNA against NADPH oxidase (NOX) 1 or 4, suggesting that ligand-activated PPARδ elicits these effects by modulating NOX-mediated ROS generation. The phosphatidylinositol 3-kinase inhibitor LY294002 also inhibited Ang II-stimulated [3H]-leucine incorporation and ROS generation by preventing membrane translocation of Rac1. These observations suggest that PPARδ is an endogenous modulator of Ang II-triggered hypertrophy of VSMCs, and is thus a potential target to treat vascular diseases associated with hypertrophic changes of VSMCs.

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<![CDATA[The planar cell polarity protein VANG-1/Vangl negatively regulates Wnt/β-catenin signaling through a Dvl dependent mechanism]]> https://www.researchpad.co/article/5c141e7ad5eed0c484d26c9a

Van Gogh-like (Vangl) and Prickle (Pk) are core components of the non-canonical Wnt planar cell polarity pathway that controls epithelial polarity and cell migration. Studies in vertebrate model systems have suggested that Vangl and Pk may also inhibit signaling through the canonical Wnt/β-catenin pathway, but the functional significance of this potential cross-talk is unclear. In the nematode C. elegans, the Q neuroblasts and their descendants migrate in opposite directions along the anteroposterior body axis. The direction of these migrations is specified by Wnt signaling, with activation of canonical Wnt signaling driving posterior migration, and non-canonical Wnt signaling anterior migration. Here, we show that the Vangl ortholog VANG-1 influences the Wnt signaling response of the Q neuroblasts by negatively regulating canonical Wnt signaling. This inhibitory activity depends on a carboxy-terminal PDZ binding motif in VANG-1 and the Dishevelled ortholog MIG-5, but is independent of the Pk ortholog PRKL-1. Moreover, using Vangl1 and Vangl2 double mutant cells, we show that a similar mechanism acts in mammalian cells. We conclude that cross-talk between VANG-1/Vangl and the canonical Wnt pathway is an evolutionarily conserved mechanism that ensures robust specification of Wnt signaling responses.

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<![CDATA[cPLA2α-/- sympathetic neurons exhibit increased membrane excitability and loss of N-Type Ca2+ current inhibition by M1 muscarinic receptor signaling]]> https://www.researchpad.co/article/5c215165d5eed0c4843f9fbb

Group IVa cytosolic phospholipase A2 (cPLA2α) mediates GPCR-stimulated arachidonic acid (AA) release from phosphatidylinositol 4,5-bisphosphate (PIP2) located in plasma membranes. We previously found in superior cervical ganglion (SCG) neurons that PLA2 activity is required for voltage-independent N-type Ca2+ (N-) current inhibition by M1 muscarinic receptors (M1Rs). These findings are at odds with an alternative model, previously observed for M-current inhibition, where PIP2 dissociation from channels and subsequent metabolism by phospholipase C suffices for current inhibition. To resolve cPLA2α’s importance, we have investigated its role in mediating voltage-independent N-current inhibition (~40%) that follows application of the muscarinic agonist oxotremorine-M (Oxo-M). Preincubation with different cPLA2α antagonists or dialyzing cPLA2α antibodies into cells minimized N-current inhibition by Oxo-M, whereas antibodies to Ca2+-independent PLA2 had no effect. Taking a genetic approach, we found that SCG neurons from cPLA2α-/- mice exhibited little N-current inhibition by Oxo-M, confirming a role for cPLA2α. In contrast, cPLA2α antibodies or the absence of cPLA2α had no effect on voltage-dependent N-current inhibition by M2/M4Rs or on M-current inhibition by M1Rs. These findings document divergent M1R signaling mediating M-current and voltage-independent N-current inhibition. Moreover, these differences suggest that cPLA2α acts locally to metabolize PIP2 intimately associated with N- but not M-channels. To determine cPLA2α’s functional importance more globally, we examined action potential firing of cPLA2α+/+ and cPLA2α-/- SCG neurons, and found decreased latency to first firing and interspike interval resulting in a doubling of firing frequency in cPLA2α-/- neurons. These unanticipated findings identify cPLA2α as a tonic regulator of neuronal membrane excitability.

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<![CDATA[Autophagic cell death associated to Sorafenib in renal cell carcinoma is mediated through Akt inhibition in an ERK1/2 independent fashion]]> https://www.researchpad.co/article/5b694661463d7e3867f4ad07

Objectives

To fully clarify the role of Mitogen Activated Protein Kinase in the therapeutic response to Sorafenib in Renal Cell Carcinoma as well as the cell death mechanism associated to this kinase inhibitor, we have evaluated the implication of several Mitogen Activated Protein Kinases in Renal Cell Carcinoma-derived cell lines.

Materials and methods

An experimental model of Renal Cell Carcinoma-derived cell lines (ACHN and 786-O cells) was evaluated in terms of viability by MTT assay, induction of apoptosis by caspase 3/7 activity, autophagy induction by LC3 lipidation, and p62 degradation and kinase activity using phospho-targeted antibodies. Knock down of ATG5 and ERK5 was performed using lentiviral vector coding specific shRNA

Results

Our data discard Extracellular Regulated Kinase 1/2 and 5 as well as p38 Mitogen Activated Protein Kinase pathways as mediators of Sorafenib toxic effect but instead indicate that the inhibitory effect is exerted through the PI3K/Akt signalling pathway. Furthermore, we demonstrate that inhibition of Akt mediates cell death associated to Sorafenib without caspase activation, and this is consistent with the induction of autophagy, as indicated by the use of pharmacological and genetic approaches.

Conclusion

The present report demonstrates that Sorafenib exerts its toxic effect through the induction of autophagy in an Akt-dependent fashion without the implication of Mitogen Activated Protein Kinase. Therefore, our data discard the use of inhibitors of the RAF-MEK-ERK1/2 signalling pathway in RCC and support the use of pro-autophagic compounds, opening new therapeutic opportunities for Renal Cell Carcinoma.

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<![CDATA[Non-Dioxin-Like Polychlorinated Biphenyls Inhibit G-Protein Coupled Receptor-Mediated Ca2+ Signaling by Blocking Store-Operated Ca2+ Entry]]> https://www.researchpad.co/article/5989da7eab0ee8fa60b99866

Polychlorinated biphenyls (PCBs) are ubiquitous pollutants which accumulate in the food chain. Recently, several molecular mechanisms by which non-dioxin-like (NDL) PCBs mediate neurodevelopmental and neurobehavioral toxicity have been elucidated. However, although the G-protein coupled receptor (GPCR) is a significant target for neurobehavioral disturbance, our understanding of the effects of PCBs on GPCR signaling remains unclear. In this study, we investigated the effects of NDL-PCBs on GPCR-mediated Ca2+ signaling in PC12 cells. We found that ortho-substituted 2,2’,6-trichlorinated biphenyl (PCB19) caused a rapid decline in the Ca2+ signaling of bradykinin, a typical Gq- and phospholipase Cβ-coupled GPCR, without any effect on its inositol 1,4,5-trisphosphate production. PCB19 reduced thapsigargin-induced sustained cytosolic Ca2+ levels, suggesting that PCB19 inhibits SOCE. The abilities of other NDL-PCBs to inhibit store-operated Ca2+ entry (SOCE) were also examined and found to be of similar potencies to that of PCB19. PCB19 also showed a manner equivalent to that of known SOCE inhibitors. PCB19-mediated SOCE inhibition was confirmed by demonstrating the ability of PCB19 to inhibit the SOCE current and thapsigargin-induced Mn2+ influx. These results imply that one of the molecular mechanism by which NDL-PCBs cause neurobehavioral disturbances involves NDL-PCB-mediated inhibition of SOCE, thereby interfering with GPCR-mediated Ca2+ signaling.

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<![CDATA[Activation of the PI3K/mTOR Pathway following PARP Inhibition in Small Cell Lung Cancer]]> https://www.researchpad.co/article/5989da3dab0ee8fa60b88a3c

Small cell lung cancer (SCLC) is an aggressive malignancy with limited treatment options. We previously found that PARP is overexpressed in SCLC and that targeting PARP reduces cell line and tumor growth in preclinical models. However, SCLC cell lines with PI3K/mTOR pathway activation were relatively less sensitive to PARP inhibition. In this study, we investigated the proteomic changes in PI3K/mTOR and other pathways that occur following PAPR inhibition and/or knockdown in vitro and in vivo. Using reverse-phase protein array, we found the proteins most significantly upregulated following treatment with the PARP inhibitors olaparib and rucaparib were in the PI3K/mTOR pathway (p-mTOR, p-AKT, and pS6) (p≤0.02). Furthermore, amongst the most significantly down-regulated proteins were LKB1 and its targets AMPK and TSC, which negatively regulate the PI3K pathway (p≤0.042). Following PARP knockdown in cell lines, phosphorylated mTOR, AKT and S6 were elevated and LKB1 signaling was diminished. Global ATP concentrations increased following PARP inhibition (p≤0.02) leading us to hypothesize that the observed increased PI3K/mTOR pathway activation following PARP inhibition results from decreased ATP usage and a subsequent decrease in stress response signaling via LKB1. Based on these results, we then investigated whether co-targeting with a PARP and PI3K inhibitor (BKM-120) would work better than either single agent alone. A majority of SCLC cell lines were sensitive to BKM-120 at clinically achievable doses, and cMYC expression was the strongest biomarker of response. At clinically achievable doses of talazoparib (the most potent PARP inhibitor in SCLC clinical testing) and BKM-120, an additive effect was observed in vitro. When tested in two SCLC animal models, a greater than additive interaction was seen (p≤0.008). The data presented here suggest that combining PARP and PI3K inhibitors enhances the effect of either agent alone in preclinical models of SCLC, warranting further investigation of such combinations in SCLC patients.

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<![CDATA[A natural product-like JAK2/STAT3 inhibitor induces apoptosis of malignant melanoma cells]]> https://www.researchpad.co/article/5989db5cab0ee8fa60be0209

The JAK2/STAT3 signaling pathway plays a critical role in tumorigenesis, and has been suggested as a potential molecular target for anti-melanoma therapeutics. However, few JAK2 inhibitors were being tested for melanoma therapy. In this study, eight amentoflavone analogues were evaluated for their activity against human malignant melanoma cells. The most potent analogue, compound 1, inhibited the phosphorylation of JAK2 and STAT3 in human melanoma cells, but had no discernible effect on total JAK2 and STAT3 levels. A cellular thermal shift assay was performed to identify that JAK2 is engaged by 1 in cell lysates. Moreover, compound 1 showed higher antiproliferative activity against human melanoma A375 cells compared to a panel of cancer and normal cell lines. Compound 1 also activated caspase-3 and cleaved PARP, which are markers of apoptosis, and suppressed the anti-apoptotic Bcl-2 level. Finally, compound 1 induced apoptosis in 80% of treated melanoma cells. To our knowledge, compound 1 is the first amentoflavone-based JAK2 inhibitor to be investigated for use as an anti-melanoma agent.

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<![CDATA[Hepatocyte nuclear factor 1α downregulates HBV gene expression and replication by activating the NF-κB signaling pathway]]> https://www.researchpad.co/article/5989db50ab0ee8fa60bdc08d

The role of hepatocyte nuclear factor 1α (HNF1α) in the regulation of gene expression and replication of hepatitis B virus (HBV) is not fully understood. Previous reports have documented the induction of the expression of viral large surface protein (LHBs) by HNF1α through activating viral Sp1 promoter. Large amount of LHBs can block the secretion of hepatitis B surface antigen (HBsAg). Here we found that HNF1α overexpression inhibited HBV gene expression and replication in Huh7 cells, resulting in marked decreases in HBsAg, hepatitis B e antigen (HBeAg) and virion productions. In contrast, knockdown of endogenous HNF1α expression enhanced viral gene expression and replication. This HNF1α-mediated inhibition did not depend on LHBs. Instead, HNF1α promoted the expression of NF-κB p65 and slowed p65 protein degradation, leading to nuclear accumulation of p65 and activation of the NF-κB signaling, which in turn inhibited HBV gene expression and replication. The inhibitor of the NF-κB signaling, IκBα-SR, could abrogate this HNF1α-mediated inhibition. While the dimerization domain of HNF1α was dispensable for the induction of LHBs expression, all the domains of HNF1α was required for the inhibition of HBV gene expression. Our findings identify a novel role of HNF1α in the regulation of HBV gene expression and replication.

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<![CDATA[Netrin-1 Reduces Monocyte and Macrophage Chemotaxis towards the Complement Component C5a]]> https://www.researchpad.co/article/5989da3bab0ee8fa60b87f58

Netrin-1, acting at its cognate receptor UNC5b, has been previously demonstrated to inhibit CC chemokine-induced immune cell migration. In line with this, we found that netrin-1 was able to inhibit CCL2-induced migration of bone marrow derived macrophages (BMDMs). However, whether netrin-1 is capable of inhibiting chemotaxis to a broader range of chemoattractants remains largely unexplored. As our initial experiments demonstrated that RAW264.7 and BMDMs expressed high levels of C5a receptor 1 (C5aR1) on their surface, we aimed to determine the effect of netrin-1 exposure on monocyte/macrophage cell migration induced by C5a, a complement peptide that plays a major role in multiple inflammatory pathologies. Treatment of RAW264.7 macrophages, BMDMs and human monocytes with netrin-1 inhibited their chemotaxis towards C5a, as measured using two different real-time methods. This inhibitory effect was found to be dependent on netrin-1 receptor signalling, as an UNC5b blocking antibody was able to reverse netrin-1 inhibition of C5a induced BMDM migration. Treatment of BMDMs with netrin-1 had no effect on C5aR1 proximal signalling events, as surface C5aR1 expression, internalisation and intracellular Ca2+ release following C5aR1 ligation remained unaffected after netrin-1 exposure. We next examined receptor distal events that occur following C5aR1 activation, but found that netrin-1 was unable to inhibit C5a induced phosphorylation of ERK1/2, Akt and p38, pathways important for cellular migration. Furthermore, netrin-1 treatment had no effect on BMDM cytoskeletal rearrangement following C5a stimulation as determined by microscopy and real-time electrical impedance sensing. Taken together these data highlight that netrin-1 inhibits monocyte and macrophage cell migration, but that the mechanism behind this effect remains unresolved. Nevertheless, netrin-1 and its cognate receptors warrant further investigation as they may represent a potential avenue for the development of novel anti-inflammatory therapeutics.

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<![CDATA[Kaempferol Inhibits Pancreatic Cancer Cell Growth and Migration through the Blockade of EGFR-Related Pathway In Vitro]]> https://www.researchpad.co/article/5989db05ab0ee8fa60bc823c

Pancreatic cancer is one of the most appalling cancers with a pessimistic prognosis. Despite many therapies, there has been no improvement of survival rates. In this study, we assessed the anti-cancer effects of kaempferol, a well known flavonoid having functional bio-activity against various malignant tumors. Kaempferol had anti-cancer effects on Miapaca-2, Panc-1, and SNU-213 human pancreatic cancer cells. In a dose-dependent manner, kaempferol decreased viability of these pancreatic cancer cells by increasing apoptosis. In particular, kaempferol effectively inhibited the migratory activity of human pancreatic cancer cells at relatively low dosages without any toxicity. The anti-cancer effect of kaempferol was mediated by inhibition of EGFR related Src, ERK1/2, and AKT pathways. These results collectively indicate that kaempferol, a phytochemical ingredient reported to have anti-viability and anti-oxidant properties, can act as a safety anti-migration reagent in human pancreatic cancer cells, which provide the rationale for further investigation of kaempferol as a strong candidate for the potential clinical trial of malignant pancreatic cancers.

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<![CDATA[Inhibition of Cardiac Kir Current (IK1) by Protein Kinase C Critically Depends on PKCβ and Kir2.2]]> https://www.researchpad.co/article/5989db4bab0ee8fa60bda490

Background

Cardiac inwardly rectifying Kir current (IK1) mediates terminal repolarisation and is critical for the stabilization of the diastolic membrane potential. Its predominant molecular basis in mammalian ventricle is heterotetrameric assembly of Kir2.1 and Kir2.2 channel subunits. It has been shown that PKC inhibition of IK1 promotes focal ventricular ectopy. However, the underlying molecular mechanism has not been fully elucidated to date.

Methods and Results

In the Xenopus oocyte expression system, we observed a pronounced PKC-induced inhibition of Kir2.2 but not Kir2.1 currents. The PKC regulation of Kir2.2 could be reproduced by an activator of conventional PKC isoforms and antagonized by pharmacological inhibition of PKCβ. In isolated ventricular cardiomyocytes (rat, mouse), pharmacological activation of conventional PKC isoforms induced a pronounced inhibition of IK1. The PKC effect in rat ventricular cardiomyocytes was markedly attenuated following co-application of a small molecule inhibitor of PKCβ. Underlining the critical role of PKCβ, the PKC-induced inhibition of IK1 was absent in homozygous PKCβ knockout-mice. After heterologous expression of Kir2.1-Kir2.2 concatemers in Xenopus oocytes, heteromeric Kir2.1/Kir2.2 currents were also inhibited following activation of PKC.

Conclusion

We conclude that inhibition of cardiac IK1 by PKC critically depends on the PKCβ isoform and Kir2.2 subunits. This regulation represents a potential novel target for the antiarrhythmic therapy of focal ventricular arrhythmias.

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<![CDATA[Cutaneous HPV8 and MmuPV1 E6 Proteins Target the NOTCH and TGF-β Tumor Suppressors to Inhibit Differentiation and Sustain Keratinocyte Proliferation]]> https://www.researchpad.co/article/5989db53ab0ee8fa60bdcee7

Cutaneous beta-papillomaviruses are associated with non-melanoma skin cancers that arise in patients who suffer from a rare genetic disorder, Epidermodysplasia verruciformis (EV) or after immunosuppression following organ transplantation. Recent studies have shown that the E6 proteins of the cancer associated beta human papillomavirus (HPV) 5 and HPV8 inhibit NOTCH and TGF-β signaling. However, it is unclear whether disruption of these pathways may contribute to cutaneous HPV pathogenesis and carcinogenesis. A recently identified papillomavirus, MmuPV1, infects laboratory mouse strains and causes cutaneous skin warts that can progress to squamous cell carcinoma. To determine whether MmuPV1 may be an appropriate model to mechanistically dissect the molecular contributions of cutaneous HPV infections to skin carcinogenesis, we investigated whether MmuPV1 E6 shares biological and biochemical activities with HPV8 E6. We report that the HPV8 and MmuPV1 E6 proteins share the ability to bind to the MAML1 and SMAD2/SMAD3 transcriptional cofactors of NOTCH and TGF-beta signaling, respectively. Moreover, we demonstrate that these cutaneous papillomavirus E6 proteins inhibit these two tumor suppressor pathways and that this ability is linked to delayed differentiation and sustained proliferation of differentiating keratinocytes. Furthermore, we demonstrate that the ability of MmuPV1 E6 to bind MAML1 is necessary for papilloma formation in experimentally infected mice. Our results, therefore, suggest that experimental MmuPV1 infection in mice will be a robust and useful experimental system to model key aspects of cutaneous HPV infection, pathogenesis and carcinogenesis.

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<![CDATA[AKT and its related molecular feature in aged mice skin]]> https://www.researchpad.co/article/5989db5dab0ee8fa60be0402

Previous studies suggest that Akt signaling promotes tissue regeneration and decreased Akt activities are found in aged tissues. However, this study finds that the expression and activation levels of Akt in the mice skin increased with age. Additionally, the expression levels of Pten, p16, p21 and p53 also elevated with increased age. Immuno-fluorescence analysis showed that Akt phosphorylation found in the epidermal cells (with increased levels of NF-κB activation) were also found. In vivo inhibition of AKT activity result in reduced NF-κB activation. Our results suggest that increasing Akt/ NF-κB is a crucial mediator of skin aging, which can increase the susceptibility of cell transformation.

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<![CDATA[Vaccinia Virus Protein C6 Inhibits Type I IFN Signalling in the Nucleus and Binds to the Transactivation Domain of STAT2]]> https://www.researchpad.co/article/5989d9ecab0ee8fa60b6cb72

The type I interferon (IFN) response is a crucial innate immune signalling pathway required for defense against viral infection. Accordingly, the great majority of mammalian viruses possess means to inhibit this important host immune response. Here we show that vaccinia virus (VACV) strain Western Reserve protein C6, is a dual function protein that inhibits the cellular response to type I IFNs in addition to its published function as an inhibitor of IRF-3 activation, thereby restricting type I IFN production from infected cells. Ectopic expression of C6 inhibits the induction of interferon stimulated genes (ISGs) in response to IFNα treatment at both the mRNA and protein level. C6 inhibits the IFNα-induced Janus kinase/signal transducer and activator of transcription (JAK/STAT) signalling pathway at a late stage, downstream of STAT1 and STAT2 phosphorylation, nuclear translocation and binding of the interferon stimulated gene factor 3 (ISGF3) complex to the interferon stimulated response element (ISRE). Mechanistically, C6 associates with the transactivation domain of STAT2 and this might explain how C6 inhibits the type I IFN signalling very late in the pathway. During virus infection C6 reduces ISRE-dependent gene expression despite the presence of the viral protein phosphatase VH1 that dephosphorylates STAT1 and STAT2. The ability of a cytoplasmic replicating virus to dampen the immune response within the nucleus, and the ability of viral immunomodulators such as C6 to inhibit multiple stages of the innate immune response by distinct mechanisms, emphasizes the intricacies of host-pathogen interactions and viral immune evasion.

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<![CDATA[Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) and Cyclic ADP-Ribose (cADPR) Mediate Ca2+ Signaling in Cardiac Hypertrophy Induced by β-Adrenergic Stimulation]]> https://www.researchpad.co/article/5989dabeab0ee8fa60bafeb2

Ca2+ signaling plays a fundamental role in cardiac hypertrophic remodeling, but the underlying mechanisms remain poorly understood. We investigated the role of Ca2+-mobilizing second messengers, NAADP and cADPR, in the cardiac hypertrophy induced by β-adrenergic stimulation by isoproterenol. Isoproterenol induced an initial Ca2+ transients followed by sustained Ca2+ rises. Inhibition of the cADPR pathway with 8-Br-cADPR abolished only the sustained Ca2+ increase, whereas inhibition of the NAADP pathway with bafilomycin-A1 abolished both rapid and sustained phases of the isoproterenol-mediated signal, indicating that the Ca2+ signal is mediated by a sequential action of NAADP and cADPR. The sequential production of NAADP and cADPR was confirmed biochemically. The isoproterenol-mediated Ca2+ increase and cADPR production, but not NAADP production, were markedly reduced in cardiomyocytes obtained from CD38 knockout mice. CD38 knockout mice were rescued from chronic isoproterenol infusion-induced myocardial hypertrophy, interstitial fibrosis, and decrease in fractional shortening and ejection fraction. Thus, our findings indicate that β-adrenergic stimulation contributes to the development of maladaptive cardiac hypertrophy via Ca2+ signaling mediated by NAADP-synthesizing enzyme and CD38 that produce NAADP and cADPR, respectively.

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<![CDATA[IL-7 Receptor Mutations and Steroid Resistance in Pediatric T cell Acute Lymphoblastic Leukemia: A Genome Sequencing Study]]> https://www.researchpad.co/article/5989da43ab0ee8fa60b8abc9

Background

Pediatric acute lymphoblastic leukemia (ALL) is the most common childhood cancer and the leading cause of cancer-related mortality in children. T cell ALL (T-ALL) represents about 15% of pediatric ALL cases and is considered a high-risk disease. T-ALL is often associated with resistance to treatment, including steroids, which are currently the cornerstone for treating ALL; moreover, initial steroid response strongly predicts survival and cure. However, the cellular mechanisms underlying steroid resistance in T-ALL patients are poorly understood. In this study, we combined various genomic datasets in order to identify candidate genetic mechanisms underlying steroid resistance in children undergoing T-ALL treatment.

Methods and Findings

We performed whole genome sequencing on paired pre-treatment (diagnostic) and post-treatment (remission) samples from 13 patients, and targeted exome sequencing of pre-treatment samples from 69 additional T-ALL patients. We then integrated mutation data with copy number data for 151 mutated genes, and this integrated dataset was tested for associations of mutations with clinical outcomes and in vitro drug response. Our analysis revealed that mutations in JAK1 and KRAS, two genes encoding components of the interleukin 7 receptor (IL7R) signaling pathway, were associated with steroid resistance and poor outcome. We then sequenced JAK1, KRAS, and other genes in this pathway, including IL7R, JAK3, NF1, NRAS, and AKT, in these 69 T-ALL patients and a further 77 T-ALL patients. We identified mutations in 32% (47/146) of patients, the majority of whom had a specific T-ALL subtype (early thymic progenitor ALL or TLX). Based on the outcomes of these patients and their prednisolone responsiveness measured in vitro, we then confirmed that these mutations were associated with both steroid resistance and poor outcome.

To explore how these mutations in IL7R signaling pathway genes cause steroid resistance and subsequent poor outcome, we expressed wild-type and mutant IL7R signaling molecules in two steroid-sensitive T-ALL cell lines (SUPT1 and P12 Ichikawa cells) using inducible lentiviral expression constructs. We found that expressing mutant IL7R, JAK1, or NRAS, or wild-type NRAS or AKT, specifically induced steroid resistance without affecting sensitivity to vincristine or L-asparaginase. In contrast, wild-type IL7R, JAK1, and JAK3, as well as mutant JAK3 and mutant AKT, had no effect. We then performed a functional study to examine the mechanisms underlying steroid resistance and found that, rather than changing the steroid receptor’s ability to activate downstream targets, steroid resistance was associated with strong activation of MEK-ERK and AKT, downstream components of the IL7R signaling pathway, thereby inducing a robust antiapoptotic response by upregulating MCL1 and BCLXL expression. Both the MEK-ERK and AKT pathways also inactivate BIM, an essential molecule for steroid-induced cell death, and inhibit GSK3B, an important regulator of proapoptotic BIM. Importantly, treating our cell lines with IL7R signaling inhibitors restored steroid sensitivity. To address clinical relevance, we treated primary T-ALL cells obtained from 11 patients with steroids either alone or in combination with IL7R signaling inhibitors; we found that including a MEK, AKT, mTOR, or dual PI3K/mTOR inhibitor strongly increased steroid-induced cell death. Therefore, combining these inhibitors with steroid treatment may enhance steroid sensitivity in patients with ALL. The main limitation of our study was the modest cohort size, owing to the very low incidence of T-ALL.

Conclusions

Using an unbiased sequencing approach, we found that specific mutations in IL7R signaling molecules underlie steroid resistance in T-ALL. Future prospective clinical studies should test the ability of inhibitors of MEK, AKT, mTOR, or PI3K/mTOR to restore or enhance steroid sensitivity and improve clinical outcome.

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