VEGF induces Tie2 shedding via a phosphoinositide 3-kinase/Akt dependent pathway to modulate Tie2 signaling

Objective— Tie2 and its ligands, the angiopoietins (Ang), are required for embryonic and postnatal angiogenesis. Previous studies have demonstrated that Tie2 is proteolytically cleaved, resulting in the production of a 75-kDa soluble receptor fragment (sTie2). We investigated mechanisms responsible for Tie2 shedding and its effects on Tie2 signaling and endothelial cellular responses. Methods and Results— sTie2 bound both Ang1 and Ang2 and inhibited angiopoietin-mediated Tie2 phosphorylation and antiapoptosis. In human umbilical vein endothelial cells, Tie2 shedding was both constitutive and induced by treatment with PMA or vascular endothelial growth factor (VEGF). Constitutive and VEGF-inducible Tie2 shedding were mediated by PI3K/Akt and p38 MAPK. Tie2 shedding was blocked by pharmacological inhibitors of either PI3K or Akt as well as by overexpression of the lipid phosphatase PTEN. In contrast, sTie2 shedding was enhanced by overexpression of either dominant negative PTEN, which increased Akt phosphorylation, or constitutively active, myristoylated Akt. Conclusions— These findings demonstrate that VEGF regulates angiopoietin-Tie2 signaling by inducing proteolytic cleavage and shedding of Tie2 via a novel PI3K/Akt-dependent pathway. These results suggest a previously unrecognized mechanism by which VEGF may inhibit vascular stabilization to promote angiogenesis and vascular remodeling.

Infection with Anaplasma phagocytophilum activates the phosphatidylinositol 3-Kinase/Akt and NF-κB survival pathways in neutrophil granulocytes

Anaplasma phagocytophilum, a Gram-negative, obligate intracellular bacterium infects primarily neutrophil granulocytes. Infection with A. phagocytophilum leads to inhibition of neutrophil apoptosis and consequently contributes to the longevity of the host cells. Previous studies demonstrated that the infection inhibits the executionary apoptotic machinery in neutrophils. However, little attempt has been made to explore which survival signals are modulated by the pathogen. The aim of the present study was to clarify whether the phosphatidylinositol 3-kinase (PI3K)/Akt and NF-?B signaling pathways, which are considered as important survival pathways in neutrophils, are involved in A. phagocytophilum-induced apoptosis delay. Our data show that infection of neutrophils with A. phagocytophilum activates the PI3K/Akt pathway and suggest that this pathway, which in turn maintains the expression of the antiapoptotic protein Mcl-1, contributes to the infection-induced apoptosis delay. In addition, the PI3K/Akt pathway is involved in the activation of NF-?B in A. phagocytophilum-infected neutrophils. Activation of NF-?B leads to the release of interleukin-8 (IL-8) from infected neutrophils, which, in an autocrine manner, delays neutrophil apoptosis. In addition, enhanced expression of the antiapoptotic protein cIAP2 was observed in A. phagocytophilum-infected neutrophils. Taken together, the data indicate that upstream of the apoptotic cascade, signaling via the PI3K/Akt pathway plays a major role for apoptosis delay in A. phagocytophilum-infected neutrophils.

Reactive Oxygen Species via Redox Signaling to PI3K/AKT Pathway Contribute to the Malignant Growth of 4-Hydroxy Estradiol-Transformed Mammary Epithelial Cells

The purpose of this study was to investigate the effects of 17-β-estradiol (E2)-induced reactive oxygen species (ROS) on the induction of mammary tumorigenesis. We found that ROS-induced by repeated exposures to 4-hydroxy-estradiol (4-OH-E2), a predominant catechol metabolite of E2, caused transformation of normal human mammary epithelial MCF-10A cells with malignant growth in nude mice. This was evident from inhibition of estrogen-induced breast tumor formation in the xenograft model by both overexpression of catalase as well as by co-treatment with Ebselen. To understand how 4-OH-E2 induces this malignant phenotype through ROS, we investigated the effects of 4-OH-E2 on redox-sensitive signal transduction pathways. During the malignant transformation process we observed that 4-OH-E2 treatment increased AKT phosphorylation through PI3K activation. The PI3K-mediated phosphorylation of AKT in 4-OH-E2-treated cells was inhibited by ROS modifiers as well as by silencing of AKT expression. RNA interference of AKT markedly inhibited 4-OH-E2-induced in vitro tumor formation. The expression of cell cycle genes, cdc2, PRC1 and PCNA and one of transcription factors that control the expression of these genes – nuclear respiratory factor-1 (NRF-1) was significantly up-regulated during the 4-OH-E2-mediated malignant transformation process. The increased expression of these genes was inhibited by ROS modifiers as well as by silencing of AKT expression. These results indicate that 4-OH-E2-induced cell transformation may be mediated, in part, through redox-sensitive AKT signal transduction pathways by up-regulating the expression of cell cycle genes cdc2, PRC1 and PCNA, and the transcription factor – NRF-1. In summary, our study has demonstrated that: (i) 4-OH-E2 is one of the main estrogen metabolites that induce mammary tumorigenesis and (ii) ROS-mediated signaling leading to the activation of PI3K/AKT pathway plays an important role in the generation of 4-OH-E2-induced malignant phenotype of breast epithelial cells. In conclusion, ROS are important signaling molecules in the development of estrogen-induced malignant breast lesions.

Activation des voies de Pl3 kinase/Akt et ERK1/2 par la ghreline et la des-acyl ghreline dans les cellules musculaires lisses vasculaires : voies potentielles de signalisation dans la modulation de l'athérosclérose

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Activation des voies de Pl3 kinase/Akt et ERK1/2 par la ghreline et la des-acyl ghreline dans les cellules musculaires lisses vasculaires : voies potentielles de signalisation dans la modulation de l'athérosclérose

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Gelsolin-mediated activation of PI3K/Akt pathway is crucial for hepatocyte growth factor-induced cell scattering in gastric carcinoma

In gastric cancer (GC), the main subtypes (diffuse and intestinal types) differ in pathological characteristics, with diffuse GC exhibiting early disseminative and invasive behaviour. A distinctive feature of diffuse GC is loss of intercellular adhesion. Although widely attributed to mutations in the CDH1 gene encoding E-cadherin, a significant percentage of diffuse GC do not harbor CDH1 mutations. We found that the expression of the actin-modulating cytoskeletal protein, gelsolin, is significantly higher in diffuse-type compared to intestinal-type GCs, using immunohistochemical and microarray analysis. Furthermore, in GCs with wild-type CDH1, gelsolin expression correlated inversely with CDH1 gene expression. Downregulating gelsolin using siRNA in GC cells enhanced intercellular adhesion and E-cadherin expression, and reduced invasive capacity. Interestingly, hepatocyte growth factor (HGF) induced increased gelsolin expression, and gelsolin was essential for HGF-medicated cell scattering and E-cadherin transcriptional repression through Snail, Twist and Zeb2. The HGF-dependent effect on E-cadherin was found to be mediated by interactions between gelsolin and PI3K-Akt signaling. This study reveals for the first time a function of gelsolin in the HGF/cMet oncogenic pathway, which leads to E-cadherin repression and cell scattering in gastric cancer. Our study highlights gelsolin as an important pro-disseminative factor contributing to the aggressive phenotype of diffuse GC.

Quantitative phosphoproteomics reveals the role of the AMPK plant ortholog SnRK1 as a metabolic master regulator under energy deprivation

Since years, research on SnRK1, the major cellular energy sensor in plants, has tried to define its role in energy signalling. However, these attempts were notoriously hampered by the lethality of a complete knockout of SnRK1. Therefore, we generated an inducible amiRNA::SnRK1α2 in a snrk1α1 knock out background (snrk1α1/α2) to abolish SnRK1 activity to understand major systemic functions of SnRK1 signalling under energy deprivation triggered by extended night treatment. We analysed the in vivo phosphoproteome, proteome and metabolome and found that activation of SnRK1 is essential for repression of high energy demanding cell processes such as protein synthesis. The most abundant effect was the constitutively high phosphorylation of ribosomal protein S6 (RPS6) in the snrk1α1/α2 mutant. RPS6 is a major target of TOR signalling and its phosphorylation correlates with translation. Further evidence for an antagonistic SnRK1 and TOR crosstalk comparable to the animal system was demonstrated by the in vivo interaction of SnRK1α1 and RAPTOR1B in the cytosol and by phosphorylation of RAPTOR1B by SnRK1α1 in kinase assays. Moreover, changed levels of phosphorylation states of several chloroplastic proteins in the snrk1α1/α2 mutant indicated an unexpected link to regulation of photosynthesis, the main energy source in plants.

The regulation of AMP-activated protein kinase by vascular endothelial growth factor

The function of the vascular endothelium is to maintain vascular homeostasis, by providing an anti-thrombotic, anti-inflammatory and vasodilatory interface between circulating blood and the vessel wall, meanwhile facilitating the selective passage of blood components such as signaling molecules and immune cells. Dysfunction of the vascular endothelium is implicated in a number of pathological states including atherosclerosis and hypertension, and is thought to precede atherogenesis by a number of years. Vascular endothelial growth factor A (VEGF) is a crucial mitogenic signaling molecule, not only essential for embryonic development, but also in the adult for regulating both physiological and pathological angiogenesis. Previous studies by our laboratory have demonstrated that VEGF-A activates AMP-activated protein kinase (AMPK), the downstream component of a signaling cascade important in the regulation of whole body and cellular energy status. Furthermore, studies in our laboratory have indicated that AMPK is essential for VEGF-A-stimulated vascular endothelial cell proliferation. AMPK activation typically stimulates anabolic processes and inhibits catabolic processes including cell proliferation, with the ultimate aim of redressing energy imbalance, and as such is an attractive therapeutic target for the treatment of obesity, metabolic syndromes, and type 2 diabetes. Metabolic diseases are associated with adverse cardiovascular outcomes and AMPK activation is reported to have beneficial effects on the vascular endothelium. The mechanism by which VEGF-A stimulates AMPK, and the functional consequences of VEGF-A-stimulated AMPK activation remain uncertain. The present study therefore aimed to identify the specific mechanism(s) by which VEGF-A regulates the activity of AMPK in endothelial cells, and how this might differ from the activation of AMPK by other agents. Furthermore, the role of AMPK in the pro-proliferative actions of VEGF-A was further examined. Human aortic and umbilical vein endothelial cells were therefore used as a model system to characterise the specific effect(s) of VEGF-A stimulation on AMPK activation. The present study reports that AMPK α1 containing AMPK complexes account for the vast majority of both basal and VEGF-A-stimulated AMPK activity. Furthermore, AMPK α1 is localized to the endoplasmic reticulum when sub-confluent, but translocated to the Golgi apparatus when cells are cultured to confluence. AMPK α2 appears to be associated with a structural cellular component, but neither α1 nor α2 complexes appear to translocate in response to VEGF-A stimulation. The present study confirms previous reports that when measured using the MTS cell proliferation assay, AMPK is required for VEGF-A-stimulated endothelial cell proliferation. However, parallel experiments measuring cell proliferation using the Real-Time Cell Analyzer xCELLigence system, do not agree with these previous reports, suggesting that AMPK may in fact be required for an aspect of mitochondrial metabolism which is enhanced by VEGF-A. Studies into the mitochondrial activity of endothelial cells have proved inconclusive at this time, but further studies into this are warranted. During previous studies in our laboratory, it was suggested that VEGF-A-stimulated AMPK activation may be mediated via the diacylglycerol (DAG)-sensitive transient receptor potential cation channel (TRPCs -3, -6 or -7) family of ion channels. The present study can neither confirm, nor exclude the expression of TRPCs in vascular endothelial cells, nor rule out their involvement in VEGF-A-stimulated AMPK activation; more specific investigative tools are required in order to characterise their involvement. Furthermore, nicotinic acid adenine dinucleotide phosphate (NAADP)-stimulated Ca2+ release from acidic intracellular organelles is not required for AMPK activation by VEGF-A. Despite what is known about the mechanisms by which AMPK is activated, far less is known concerning the downregulation of AMPK activity, as observed in human and animal models of metabolic disease. Phosphorylation of AMPK α1 Ser485 (α2 Ser491) has recently been characterised as a mechanism by which the activity of AMPK is negatively regulated. We report here for the first time that VEGF-A stimulates AMPK α1 Ser485 phosphorylation independently of the previously reported AMPK α1 Ser485 kinases Akt (protein kinase B) and ERK1/2 (extracellular signal-regulated kinase 1/2). Furthermore, inhibition of protein kinase C (PKC), the activity of which is reported to be elevated in metabolic disease, attenuates VEGF-A- and phorbol 12-myristate 13-acetate (PMA)-stimulated AMPK α1 Ser485 phosphorylation, and increases basal AMPK activity. In contrast to this, PKC activation reduces AMPK activity in human vascular endothelial cells. Attempts to identify the PKC isoform responsible for inhibiting AMPK activity suggest that it is one (or more) of the Ca2+-regulated DAG-sensitive isoforms of PKC, however cross regulation of PKC isoform expression has limited the present study. Furthermore, AMPK α1 Ser485 phosphorylation was inversely correlated with human muscle insulin sensitivity. As such, enhanced AMPK α1 Ser485 phosphorylation, potentially mediated by increased PKC activation may help explain some of the reduced AMPK activity observed in metabolic disease.

Low-Molecular-Weight Fucoidan Induces Endothelial Cell Migration via the PI3K/AKT Pathway and Modulates the Transcription of Genes Involved in Angiogenesis

Low-molecular-weight fucoidan (LMWF) is a sulfated polysaccharide extracted from brown seaweed that presents antithrombotic and pro-angiogenic properties. However, its mechanism of action is not well-characterized. Here, we studied the effects of LMWF on cell signaling and whole genome expression in human umbilical vein endothelial cells and endothelial colony forming cells. We observed that LMWF and vascular endothelial growth factor had synergistic effects on cell signaling, and more interestingly that LMWF by itself, in the absence of other growth factors, was able to trigger the activation of the PI3K/AKT pathway, which plays a crucial role in angiogenesis and vasculogenesis. We also observed that the effects of LMWF on cell migration were PI3K/AKT-dependent and that LMWF modulated the expression of genes involved at different levels of the neovessel formation process, such as cell migration and cytoskeleton organization, cell mobilization and homing. This provides a better understanding of LMWF's mechanism of action and confirms that it could be an interesting therapeutic approach for vascular repair.

News telewizyjny jako akt retoryczny

Artykuł przedstawia news telewizyjny jako akt retoryczny. News telewizyjny rozumiany jest jako relacja o wydarzeniu zbudowana zazwyczaj z zapowiedzi i krótkiego kilkuminutowego materiału filmowego (nazywanego czasem felietonem), będącego rezultatem pracy reporter-skiej, wyemitowana w telewizji, zazwyczaj jako jeden z elementów w strukturze magazynu informacyjnego, ale również jako element bloków programowych telewizji informacyjnych. Newsy telewizyjne funkcjonują w mediach jako jeden z elementów działań strategicznych nadawców medialnych nastawionych na określone cele, w tym cele wizerunkowe, polityczne i ekonomiczne. Newsy powstają jako reakcja na konkretne wydarzenia, zaspokajają potrzebę informacji, ale służą też kreowaniu obrazu świata, skupianiu uwagi na pewnych aspektach rze-czywistości, wartościowaniu jej, przekonywaniu o ważności lub nieważności pewnych zda-rzeń. Pełnią funkcje informacyjne, ale też kreacyjne, wzorcotwórcze, stymulacyjne, wartościu-jące, i w końcu estetyczne. Dla osiągnięcia tych celów są odpowiednio konstruowane, z uwzględnieniem specyfiki i potrzeb odbiorcy oraz ethosu nadawcy. W tym sensie są więc ak-tami „retorycznymi”, a więc zamierzonymi, celowymi, będącymi odpowiedzią na określoną sytuację (uwarunkowane są historycznie, społecznie i politycznie) i adresowanymi do określo-nych odbiorców.

Glosa do postanowienia Naczelnego Sądu Administracyjnego z dnia 13 stycznia 2016 r., sygn. akt II GZ 927/15

W glosowanym orzeczeniu Naczelny Sąd Administracyjny stwierdził, że od postanowień wojewódzkich sądów administracyjnych odrzucających skargi z przyczyn formalnych w sprawach wszczętych przed dniem wejścia w życie nowelizacji Prawa o postępowaniu przed sądami administracyjnymi (co miało miejsce 15 sierpnia 2015 r.) przysługuje zażalenie, a nie skarga kasacyjna. Przedmiotowe zagadnienie prawne sprowadza się do właściwej wykładni źle sformułowanych przepisów intertemporalnych. W glosie zaprezentowano istotę środków odwoławczych w postępowaniu sądowoadministracyjnym i scharakteryzowano krótko ostatnią nowelizację Prawa o postępowaniu przed sądami administracyjnymi. Autor prezentuje stanowisko krytyczne w stosunku do tezy postanowienia Naczelnego Sądu Administracyjnego, wskazując, że sąd powinien rozstrzygnąć sprawę co do jej istoty. Pogląd ten został poparty odpowiednią argumentacją z uwzględnieniem konkurencyjnego orzecznictwa Naczelnego Sądu Administracyjnego.

Pochodzenie życia - wyjątkowy akt samorództwa?

Książka skierowana jest przede wszystkim dla adeptów kierunków: geologii i biologii a jako lektura uzupełniająca może być wykorzystany także w paleontologii i geologii historycznej. W zamierzeniu autora praca ta ma dostarczyć niezbędnej wiedzy na temat procesów towarzyszących początkom życia na naszej planecie. Opracowanie to jest pierwszą na polskim rynku wydawniczym próbą syntetyzowania najistotniejszych koncepcji związanych z pochodzeniem życia na Ziemi. Dzięki klarownemu układowi pomaga przyswoić treści „rozproszone” w innych publikacjach, dając tym samym niezbędne podstawy do studiów paleontologicznych. Natomiast starannie dobrana bibliografia pozwala odpowiednio poszerzyć interesujące odbiorcę wątki. Język książki, bardzo przystępny, nie jest jednak zbyt eseistyczny. Z jednej strony wyróżnia to tę pozycję spośród innych publikacji o charakterze popularyzatorskim, nie zawsze solidnie traktujących kwestie merytoryczne. Z drugiej powinno ułatwić przyswojenie tych partii wiedzy, które stereotypowo traktuje się jako „trudne”. Całość stanowi zatem lekturę przystępną nawet dla tych czytelników którzy pozostając poza akademickim trybem, interesują się studiami z zakresu nauk przyrodniczych i geologicznych.

Regulation of prostate cancer cell function by activators of AMP-activated protein kinase

AMP-activated protein kinase (AMPK) is a key regulator of cell energy homeostasis. More recently, it has become apparent that AMPK regulates cell proliferation, migration and inflammation. Previous evidence has suggested that AMPK may influence proliferation and invasion by regulating the pro-proliferative mitogen-activated protein kinases (MAPKs). However, the mechanisms underlying this crosstalk between AMPK and MAPK signalling are not fully understood. As AMPK activation has been reported to have anti-proliferative effects, there has been increasing interest in AMPK activation as a therapeutic target for tumourigenesis. The aim of this study was to investigate whether AMPK activation influenced prostate cancer (PC) cell line proliferation, migration and signalling. Therefore, different PC cell lines were incubated with two structurally-unrelated molecules that activate AMPK by different mechanisms, AICAR and A769662. Both chemicals activated AMPK in a concentration- and time-dependent manner in PC3, DU145 and LNCaP cell lines. AMPK activity as assessed by AMPK activating phosphorylation as well as phosphorylation of the AMPK substrate ACC increased along with tumour severity in PC biopsies. Furthermore, both activators of AMPK decreased cell proliferation and migration in the androgen-independent PC cell lines PC3 and DU145. Inhibition of proliferation by A769662 was attenuated in AMPK α1-/- AMPK α2-/- knockout (KO) mouse embryonic fibroblasts (MEFs) compared to wild type (WT) MEFs, and the inhibitory effect on migration of AICAR lost significance in PC3 cells infected with adenoviruses expressing a dominant negative AMPK α mutant, indicating these effects are partially mediated by AMPK. Furthermore, long-term activation of AMPK was associated with inhibition of both the phosphatidylinositol 3’-kinase/protein kinase B (PI3K/Akt) signalling pathway in addition to the extracellular signal-regulated kinase 1/2 (ERK1/2) signalling pathway. Indeed, the actions of AMPK activators on PC cell line viability were mimicked by selective inhibitors of Akt and ERK1/2 pathways. In contrast to the effects of prolonged incubation with AMPK activators, short-term incubation with AMPK activators had no effect on epidermal growth factor (EGF)-stimulated ERK1/2 phosphorylation in PC cell lines. In addition, AMPK activation did not influence phosphorylation of the other MAPK family members p38 and JNK. Interestingly, both AICAR and A769662 decreased EGF-stimulated ERK5 phosphorylation in PC3, DU145 and LNCaP cells as assessed with an anti-phospho-ERK5 antibody. Further characterisation of this effect indicated that prior stimulation with the AMPK activators had no effect on ERK5 phosphorylation stimulated by transient transfection with a constitutively active ERK5 kinase (MEK5DD), which represents the only known canonical kinase for ERK5. Intriguingly, the pattern of EGF-stimulated ERK5 phosphorylation was distinct from that mediated by MEK5DD activation of ERK5. This finding indicates that AMPK activation inhibits EGF-stimulated ERK5 phosphorylation at a point at or above the level of MEK5, although why EGF and constitutively active MEK5 stimulate markedly different immunoreactive species recognised by the anti-phospho-ERK5 antibody requires further study. A769662 had a tendency to reduce EGF-stimulated ERK5 phosphorylation in WT MEFs, yet was without effect in MEFs lacking AMPK. These data indicate that AMPK may underlie the effect of A769662 to reduce EGF-stimulated ERK5 phosphorylation. Prolonged stimulation of PC cell lines with AICAR or A769662 inhibited EGF-stimulated Akt Ser473 phosphorylation, whereas only incubation with A769662 rapidly inhibited Akt phosphorylation. This difference in the actions of the different AMPK activators may suggest an AMPK-independent effect of A769662. Furthermore, AICAR increased phosphorylation of Akt in WT MEFs, an effect that was absent in MEFs lacking AMPK, indicating that this effect of AICAR may be AMPK-dependent. Taken together, the data presented in this study suggest that AMPK activators markedly inhibit proliferation and migration of PC cell lines, reduce EGF-stimulated ERK1/2 and Akt phosphorylation after prolonged incubation and rapidly inhibit ERK5 phosphorylation. Both AMPK activators exhibit a number of effects that are likely to be independent of AMPK in PC cell lines, although inhibition of ERK1/2, ERK5 and Akt may underlie the effects of AMPK activators on proliferation, viability and migration. Further studies are required to understand the crosstalk between those signalling pathways and their underlying significance in PC progression.