IGF-1 increases invasive potential of MCF 7 breast cancer cells and induces activation of latent TGF-β1 resulting in epithelial to mesenchymal transition

<p>Abstract</p> <p>Introduction</p> <p>TGF-β signaling has been extensively studied in many developmental contexts, amongst which is its ability to induce epithelial to mesenchymal transitions (EMT). EMTs play crucial roles during embryonic development and have also come under intense scrutiny as a mechanism through which breast cancers progress to become metastatic. Interestingly, while the molecular hallmarks of EMT progression (loss of cell adhesion, nuclear localization of β-catenin) are straightforward, the cellular signaling cascades that result in an EMT are numerous and diverse. Furthermore, most studies describing the biological effects of TGF-β have been performed using high concentrations of active, soluble TGF-β, despite the fact that TGF-β is produced and secreted as a latent complex.</p> <p>Methods</p> <p>MCF-7 breast cancer cells treated with recombinant IGF-1 were assayed for metalloproteinase activity and invasiveness through a matrigel coated transwell invasion chamber. IGF-1 treatments were then followed by the addition of latent-TGF-β1 to determine if elevated levels of IGF-1 together with latent-TGF-β1 could cause EMT.</p> <p>Results</p> <p>Results showed that IGF-1 - a molecule known to be elevated in breast cancer is a regulator of matrix metalloproteinase activity (MMP) and the invasive potential of MCF-7 breast cancer cells. The effects of IGF-1 appear to be mediated through signals transduced via the PI3K and MAPK pathways. In addition, increased IGF-1, together with latent TGF-β1 and active MMPs result in EMT.</p> <p>Conclusions</p> <p>Taken together our data suggest a novel a link between IGF-1 levels, MMP activity, TGF-β signaling, and EMT in breast cancer cells.</p

Analysis of the MMP-dependent and independent functions of tissue inhibitor of metalloproteinase-2 on the invasiveness of breast cancer cells

Matrix metalloproteinases (MMPs) are secreted endopeptidases that play an essential role in remodeling the extracellular matrix (ECM). MMPs are primarily active during development, when the majority of ECM remodeling events occurs. In adults, elevated MMP activity has been observed in many pathological conditions such as cancer and osteoarthritis. The proteolytic activity of MMPs is controlled by their natural inhibitors - the tissue inhibitor of metalloproteinases (TIMPs). In addition to blocking MMP-mediated proteolysis, TIMPs have a number of MMP-independent functions including binding to cell surface proteins thereby stimulating signaling cascades. TIMP-2, the most studied member of the family, can both inhibit and activate MMPs directly, as well as inhibit MMP activity indirectly by upregulating expression of RECK, a membrane anchored MMP regulator. While TIMP-2 has been shown to play important roles in breast cancer, we describe how the MMP-independent effects of TIMP-2 can modulate the invasiveness of MCF-7, T47D and MDA-MB-231 breast cancer cells. Using an ALA + TIMP-2 mutant which is devoid of MMP inhibition, but still capable of initiating specific cell signaling cascades, we show that TIMP-2 can differentially affect MMP activity and cellular invasiveness in both an MMP dependent and independent manner. More specifically, MMP activity and invasiveness is increased with the addition of exogenous TIMP-2 in poorly invasive cell lines whereas it is decreased in highly invasive cells lines (MDA-MB-231). Conversely, the addition of ALA + TIMP-2 resulted in decreased invasiveness regardless of cell line. © The International CCN Society 2012

Adaptation of a mouse Doppler echocardiograph system for assessing cardiac function and thermal performance in a juvenile salmonid

Measures of cardiac performance are pertinent to the study of thermal physiology and exercise in teleosts, particularly as they pertain to migration success. Increased heart rate, stroke volume and cardiac output have previously been linked to improved swimming performance and increased upper thermal tolerance in anadromous salmonids. To assess thermal performance in fishes, it has become commonplace to measure the response of maximum heart rate to warming using electrocardiograms. However, electrocardiograms do not provide insight into the hemodynamic characteristics of heart function that can impact whole-animal performance. Doppler echocardiography is a popular tool used to examine live animal processes, including real-time cardiac function. This method allows for nonsurgical measurements of blood flow velocity through the heart and has been used to detect abnormalities in cardiovascular function, particularly in mammals. Here, we show how a mouse Doppler echocardiograph system can be adapted for use in a juvenile salmonid over a range of temperatures and timeframes. Using this compact, noninvasive system, we measured maximum heart rate, atrioventricular (AV) blood flow velocity, the early flow-atrial flow ratio and stroke distance in juvenile Atlantic salmon (Salmo salar) during acute warming. Using histologically determined measures of AV valve area, we show how stroke distance measurements obtained with this system can be used to calculate ventricular inflow volume and approximate cardiac output. Further, we show how this Doppler system can be used to determine cardiorespiratory thresholds for thermal performance, which are increasingly being used to predict the consequences that warming water temperatures will have on migratory fishes

The state of Democracy in the Republic of Macedonia – Freefall or a Temporary Crisis?

Demokratizacija tranzicionih društava nije ni linearan ni ravnomeran proces. Usponi i padovi u razvoju demokratije povezani su kako sa internim tako i sa eksternim faktorima koji utiču na određeno društvo koje se nalazi u procesu demokratske tranzicije i konsolidacije. Republika Makedonija ni u kom smislu nije izuzetak iz ovog pravila. Naime, analiza poslednjih pet godina razvitka makedonskog društva i političke scene jasno ukazuje na to da je stanje demokratije u makedonskom društvu u krizi, a tek treba da se vidi da li će ta kriza biti privremenog ili trajnijeg karaktera. S jedne strane, građansko društvo u Makedoniji pokazuje zabrinjavajući konfliktni kapacitet. Iako su ovi sukobi do sada bili pretežno etničke prirode, oni sve više postaju i ideološki. S druge strane, opštoj konfliktnosti makedonskog društva doprinosi i nezrelost partijskog sistema, koji se nakon decembarskih događaja 2012. nalazi u najtežoj krizi posle konflikta 2001. godine. Kad se ovoj konstelaciji doda i kompletna blokada procesa evroatlantskih integracija, jasno je da se Republika Makedonija nalazi u vrlo nepovoljnom političkom trenutku. Ova analiza ima za cilj ne samo da razjasni trenutno stanje po navedenim pitanjima, već i da ukaže na idiosinkretičnost makedonskog demokratskog razvoja, koji je izgleda potpuno drugačiji u odnosu na stanje u regionu.The state of democracy in the Republic of Macedonia in the last five years indicates a serious crisis of democracy which is hard to qualify. Located anywhere between a “free fall” and a “temporary crisis” the state of democracy in Macedonia is undergoing a challenging period on many accounts. On one hand, a simple analysis of civil society indicates that the political basis of the country has rarely been more conflictual, not just along ethnic, but also along ideological lines, especially in the Macedonian ethnic block. Even more so, the political party scene also undergoes its possibly worst period, culminating in the December 24th 2012 events in the Macedonian Parliament, a display of political violence that has completely derogated procedural and parliamentary democracy in Macedonia, and has put the country in the biggest crisis ever since the conflict in 2001. If one adds up the completely blocked EU perspectives of the country, mostly due to the unresolved name dispute with neighboring Greece, that it becomes clear that the Republic of Macedonia is a very unfavorable position, or as benchmarks of many international organizations show in a serious democratic descend

Inhibition of MT1-MMP proteolytic function and ERK1/2 signalling influences cell migration and invasion through changes in MMP-2 and MMP-9 levels

Membrane type-1 matrix metalloproteinase (MT1-MMP, MMP-14) is a unique protease that cleaves extracellular proteins, activates proMMPs, and initiates intracellular signalling. MCF-7 cells are non-invasive and deficient in MT1-MMP, MMP-2, and MMP-9 expression. We created an MCF-7 cell line (C2) that stably produces active MT1-MMP and demonstrated increased ERK1/2 phosphorylation. MAPK inhibition in this cell line showed an inverse relationship in MMP-2 and MMP-9 transcripts where levels of these genes increased and decreased, respectively. Using invasive MDA-MB 231 cells that endogenously produce MT1-MMP and have naturally high pERK levels, we demonstrated the identical inverse relationship between MMP-2 and -9 transcript and protein levels, suggesting that this novel relationship is conserved amongst MT1-MMP positive breast cancer cells. To further analyze the relationship between MMP-2 and -9 levels, we chemically inhibited activation and catalytic activity of MT1-MMP using a furin and MMP inhibitor, respectively, to show that interference with the functions of MT1-MMP induced changes in MMP-2 and 9 transcript levels that were always inverse of each other, and likely mediated by differential transcriptional activity of the NF-κB transcription factor. Furthermore, we analyzed the functional consequences of these expression changes to show MMP, and in particular ERK, inhibition decreased migration and invasion using 2D culture, and inhibits the formation of an invasive phenotype in Matrigel 3D culture. This study demonstrated a novel inverse transcriptional relationship between MMP-2 and -9 levels and MT1-MMP activity that have functional consequences, and also showed that increases in the levels of MMPs does not necessarily correlate with an invasive phenotype

PEX11β induces peroxisomal gene expression and alters peroxisome number during early Xenopus laevis development

<p>Abstract</p> <p>Background</p> <p>Peroxisomes are organelles whose roles in fatty acid metabolism and reactive oxygen species elimination have contributed much attention in understanding their origin and biogenesis. Many studies have shown that <it>de novo </it>peroxisome biogenesis is an important regulatory process, while yeast studies suggest that total peroxisome numbers are in part regulated by proteins such as Pex11, which can facilitate the division of existing peroxisomes. Although <it>de novo </it>biogenesis and divisions are likely important mechanisms, the regulation of peroxisome numbers during embryonic development is poorly understood. Peroxisome number and function are particularly crucial in oviparous animals such as frogs where large embryonic yolk and fatty acid stores must be quickly metabolized, and resulting reactive oxygen species eliminated. Here we elucidate the role of Pex11β in regulating peroxisomal gene expression and number in <it>Xenopus laevis </it>embryogenesis.</p> <p>Results</p> <p>Microinjecting haemagglutinin (HA) tagged Pex11β in early embryos resulted in increased RNA levels for peroxisome related genes PMP70 and catalase at developmental stages 10 and 20, versus uninjected embryos. Catalase and PMP70 proteins were found in punctate structures at stage 20 in control embryos, whereas the injection of ectopic HA-Pex11β induced their earlier localization in punctate structures at stage 10. Furthermore, the peroxisomal marker GFP-SKL, which was found localized as peroxisome-like structures at stage 20, was similarly found at stage 10 when co-microinjected with HA-Pex11β.</p> <p>Conclusions</p> <p>Overexpressed Pex11β altered peroxisomal gene levels and induced the early formation of peroxisomes-like structures during development, both of which demonstrate that Pex11β may be a key regulator of peroxisome number in early Xenopus embryos.</p

Modulation of RECK levels in Xenopus A6 cells: effects on MT1-MMP, MMP-2 and pERK levels

Background: MT1-MMP is a cell-surface enzyme whose regulation of pro-MMP-2 and ERK activation position it as a key facilitator of ECM remodelling and cell migration. These processes are modulated by endogenous MMP inhibitors, such as RECK, a GPI-anchored protein which has been shown to inhibit both MT1-MMP and MMP-2 activity. Our previous studies have revealed a link between MT1-MMP levels, and pro-MMP-2 and ERK activation in mammalian cells, as well as MT1-MMP and RECK co-localization in Xenopus embryos. We here investigated how modulation of RECK would impact MT1-MMP and MMP-2 levels, as well as ERK signalling in Xenopus A6 cells. Results: We used a Morpholino approach to knockdown RECK, plasmid transfection to overexpress RECK, and PI-PLC treatment to shed RECK from the cell surface of Xenopus A6 cells. RECK reduction did not alter pERK or MT1-MMP levels, nor MMP-2 activity as measured by zymography; thus RECK-knockdown cells maintained the ability to remodel the ECM. RECK overexpression and PI-PLC treatment both increased ECM remodelling potential through increased MT1-MMP protein and relative MMP-2 activation levels. Conclusions: RECK changes that reduce the ability of the cell to remodel the ECM (overexpression and cell surface shedding) are compensated for by increases in MT1-MMP, and MMP-2 levels as seen by zymography

Revolucija koja je pojela svoju decu: Šarena revolucija od konsenzusa do razdora

The main goal of this essay is to provide an in-depth analysis of the trajectory of the Colourful Revolution (CR) in North Macedonia as a social movement. From a more general perspective, the paper engages with the growing interest in the literature that explores the correlation between social movements and democratisation processes, especially in societies that fall into the category of hybrid regimes. The Colourful Revolution is a good example of a protest movement that has created effective regime change. It presented a complex social movement encompassing many fragmented social and political groups gathered around the idea of a common adversary. Additionally, the Colourful Revolution has one particularity: it is a social movement that has undergone a full developmental circle – formation through utilization of political opportunity frameworks, a period of activity and success and dissolution. Drawing on literature of the political process, opportunity frameworks and cycles of social movements, the paper argues that social movements such as the Colourful Revolution are not just temporary and unstable structures but are also highly dependent on the existence of a common target of the social activism in question. The removal from power of political actors that have been the reason for mobilisation of a complex and diverse network of social and political activism resulted in an absence of an adhesive factor holding together all the parts of this complex system. The absence initiated gradual discord and dissolution of different factions within the social movement (CR in this case) and reveals its true nature – temporary, ideologically diverse, conflictual, and even undemocratic in some respects.Glavni cilj ovog rada jeste da pruži detaljnu analizu putanje Šarene revolucije (ŠR) u Severnoj Makedoniji kao društvenog pokreta. Iz opštije perspektive, rad se bavi rastućim interesovanjem za literaturu koja istražuje korelaciju između društvenih pokreta i procesa demokratizacije, pogotovo u društvima koja spadaju u kategoriju hibridnih režima. Šarena revolucija predstavlja dobar primer protestnog pokreta koji je stvorio efektivnu promenu režima. Predstavljala je, tačnije, složen društveni pokret koji je obuhvatio mnoge fragmentirane društvene i političke grupe koje su se okupile oko ideje postojanja zajedničkog protivnika. Povrh ovoga, Šarena revolucija ima jednu posebnost. To je, naime, društveni pokret koji je prošao pun razvojni krug: formiranje kroz korišćenje političkih prilika, period aktivnosti i uspeha, te period raspada. Oslanjajući se na literaturu o političkom procesu, okvirima mogućnosti i ciklusima društvenih pokreta, u ovom radu se tvrdi da društveni pokreti poput Šarene revolucije nisu samo privremene i nestabilne strukture, već da oni u značajnoj meri zavise od postojanja zajedničke mete društvenog aktivizma. Uklanjanje sa vlasti političkih aktera koji su bili razlog za mobilizaciju složene i raznolike mreže društvenog i političkog aktivizma rezultiralo je odsustvom spajajućeg faktora koji drži na okupu sve delove ovog složenog sistema. Ovo odsustvo pokrenulo je postepeni razdor i raspadanje različitih frakcija unutar društvenog pokreta (u ovom slučaju ŠR), te je otkrilo njegovu pravu prirodu – privremenu, ideološki raznoliku, konfliktnu, pa čak i nedemokratsku u nekim aspektima

Stable expression of α1-antitrypsin Portland in MDA-MB-231 cells increased MT1-MMP and MMP-9 levels, but reduced tumour progression.

The membrane bound matrix metalloproteinase MT1-MMP plays roles in modulating cell movement, independent of its abilities to remodel the extracellular matrix. Unlike many MMPs, MT1-MMP is activated in the Golgi prior to secretion by a pro-protein convertase, primarily furin. Regulation of the activation of pro-MT1-MMP has been methodically investigated, as altering the level of the active protein has broad implications in both activating other proMMPs, including pro-MMP-2, and many subsequent remodelling events. Our previous work in MCF-7 cells has demonstrated that modest, and not extremely high, levels of active MT1-MMP manifests into altered cell morphology and movement. At this low but optimal amount of MT1-MMP protein, changes to MT1-MMP levels are always mirrored by MMP-9 and pERK levels, and always opposite to MMP-2 levels. In this study, stable expression of the furin inhibitor α1- antitrypsin Portland (α1-PDX) in MDA-MB-231 cells increased overall MT1-MMP levels, but cells maintained a 21% proportion of pro-MT1-MMP. The increase in MT1- MMP was mirrored by increases in MMP-9 and pERK, but a decrease in MMP-2. These changes were associated with increased NF-κB transcription. In vitro analysis showed that α1-PDX decreased cell protrusions and migration, and this manifested as decreased tumourigenesis when examined in vivo using a chick CAM assay