Arginase activity in a murine macrophage cell line (RAW264.7) stimulated with lipopolysaccharide from Actinobacillus actinomycetemcomitans

Aims: The aim of the present study was to determine the role of cyclic adenosine monophosphate (cAMP) on arginase activity in a murine macrophage cell line (RAW264.7 cells) stimulated with lipopolysaccharide (LPS) from Actinobacillus actinomycetemcomitans. Materials and methods: The cells were treated with A. actinomycetemcomitans LPS for 24 h. The effects of SQ22536 (an adenylyl cyclase inhibitor), ODQ (a guanylyl cyclase inhibitor), dibutyryl cAMP (a cAMP analog), 8-bromo cyclic guanosine monophosphate (a cGMP analog), forskolin (an adenylyl cylase activator), and cycloheximide (a protein synthesis inhibitor) on arginase activity in A. actinomycetemcomitans LPS-stimulated RAW264.7 cells were also determined. Arginase activity was assessed in LPS-stimulated cells in the presence of 3-isobutyl-1-methylxanthine (IBMX), siguazodan and rolipram [phosphodiesterase (PDE) inhibitors] as well as KT5720 [a protein kinase A (PKA) inhibitor]. Results: Arginase activity in A. actinomycetemcomitans LPS-stimulated RAW264.7 cells was suppressed by SQ22536 but not ODQ. Enhancement of arginase activity was observed in the presence of cAMP analog or forskolin but not cGMP analog. Cycloheximide blocked arginase activity in the cells in the presence of cAMP analog or forskolin with or without A. actinomycetemcomitans LPS. IBMX augmented arginase activity in A. actinomycetemcomitans LPS-stimulated cells. Rolipram (a PDE4 inhibitor) increased the levels of arginase activity higher than siguazodan (a PDE3 inhibitor) in the antigen-stimulated cells. The effect of cAMP analog or forskolin on arginase activity in the presence or absence of A. actinomycetemcomitans LPS was blocked by the PKA inhibitor (KT5720). Conclusion: The results of the present study suggest that A. actinomycetemcomitans LPS may stimulate arginase activity in murine macrophages (RAW264.7 cells) in a cAMP-PKA-dependent pathway.

Fatty acid and peptide profiles in plasma membrane and membrane rafts of PUFA supplemented RAW264.7 macrophages

The eukaryotic cell membrane possesses numerous complex functions, which are essential for life. At this, the composition and the structure of the lipid bilayer are of particular importance. Polyunsaturated fatty acids may modulate the physical properties of biological membranes via alteration of membrane lipid composition affecting numerous physiological processes, e.g. in the immune system. In this systematic study we present fatty acid and peptide profiles of cell membrane and membrane rafts of murine macrophages that have been supplemented with saturated fatty acids as well as PUFAs from the n-3, the n-6 and the n-9 family. Using fatty acid composition analysis and mass spectrometry-based peptidome profiling we found that PUFAs from both the n-3 and the n-6 family have an impact on lipid and protein composition of plasma membrane and membrane rafts in a similar manner. In addition, we found a relation between the number of bis-allyl-methylene positions of the PUFA added and the unsaturation index of plasma membrane as well as membrane rafts of supplemented cells. With regard to the proposed significance of lipid microdomains for disease development and treatment our study will help to achieve a targeted dietary modulation of immune cell lipid bilayers.

Role of nuclear factor kappa B and reactive oxygen species in the tumor necrosis factor-a-induced epithelial-mesenchymal transition of MCF-7 cells

The microenvironment of the tumor plays an important role in facilitating cancer progression and activating dormant cancer cells. Most tumors are infiltrated with inflammatory cells which secrete cytokines such as tumor necrosis factor-a (TNF-a). To evaluate the role of TNF-a in the development of cancer we studied its effects on cell migration with a migration assay. The migrating cell number in TNF-a -treated group is about 2-fold of that of the control group. Accordingly, the expression of E-cadherin was decreased and the expression of vimentin was increased upon TNF-a treatment. These results showed that TNF-a can promote epithelial-mesenchymal transition (EMT) of MCF-7 cells. Further, we found that the expression of Snail, an important transcription factor in EMT, was increased in this process, which is inhibited by the nuclear factor kappa B (NFkB) inhibitor aspirin while not affected by the reactive oxygen species (ROS) scavenger N-acetyl cysteine. Consistently, specific inhibition of NFkB by the mutant IkBa also blocked the TNF-a-induced upregulation of Snail promoter activity. Thus, the activation of NFkB, which causes an increase in the expression of the transcription factor Snail is essential in the TNF-a-induced EMT. ROS caused by TNF-a seemed to play a minor role in the TNF-a-induced EMT of MCF-7 cells, though ROS per se can promote EMT. These findings suggest that different mechanisms might be responsible for TNF-a - and ROS-induced EMT, indicating the need for different strategies for the prevention of tumor metastasis induced by different stimuli.

Cell death and lumen formation in spheroids of MCF-7 cells

3D (three-dimensional) cell culture permits a more integrated analysis of the relationship between cells, inserting them into a structure more closely resembling the cellular microenvironment in vivo. The development of in vitro parameters to approximate in vivo 3D cellular environments makes a less reductionist interpretation of cell biology possible. For breast cells, in vitro 3D culture has proven to be an important tool for the analysis of luminal morphogenesis. A greater understanding of this process is necessary because alterations in the lumen arrangement are associated with carcinogenesis. Following lumen formation in 3D cell culture using laser scanning confocal microscopy, we observed alterations in the arrangement of cytoskeletal components (F-actin and microtubules) and increasing levels of cell death associated with lumen formation. The formation of a polarized monolayer facing the lumen was characterized through 3D reconstructions and the use of TEM (transmission electron microscopy), and this process was found to occur through the gradual clearing of cells from the medullary region of the spheroids. This process was associated with different types of cell death, such as apoptosis, autophagy and entosis. The present study showed that changes in the extracellular matrix associated with long periods of time in 3D cell culture lead to the formation of a lumen in MCF-7 cell spheroids and that features of differentiation such as lumen and budding formation occur after long periods in 3D culture, even in the absence of exogenous extracellular compounds.

The effect of overexpression of the protein tyrosine phosphatase PTPMEG on cell growth and on colony formation in soft agar in COS-7 cells

We established stable COS-7 cell lines overexpressing recombinant PTPMEG and an inactive mutant form in which the active site cysteine is mutated to serine (PTPMEGCS). We found that both endogenous and recombinant enzyme were primarily located in the membrane and cytoskeletal fractions of COS-7 cells. Endogenous PTPMEG accounts for only 1/3000th of the total tyrosine phosphatase activity in COS-7 cells and transfected cells expressed 2- to 7-fold higher levels of the enzyme. These levels of overexpression did not result in detectable changes in either total tyrosine phosphatase activity or the state of protein tyrosine phosphorylation as determined by immunoblotting of cell homogenates with anti-phosphotyrosine antibodies. Despite the low levels of activity for PTPMEG, we found that overexpressing cells grew slower and reached confluence at a lower density than vector transfected cells. Surprisingly, PTPMEGCS-transfected cells also reach confluence at a lower density than vector-transfected cells, although they grow to higher density than PTPMEG-transfected cells. Both constructs inhibited the ability of COS-7 cells to form colonies in soft agar, with the native PTPMEG having a greater effect (30-fold) than PTPMEGCS (10-fold). These results indicate that in COS-7 cells both PTPMEG and PTPMEGCS inhibit cell proliferation, reduce the saturation density, and block the ability of these cells to grow without adhering to a solid matrix.

Hereditary hemochromatosis: Effects of C282Y and H63D mutations on association with β2-microglobulin, intracellular processing, and cell surface expression of the HFE protein in COS-7 cells

Hereditary hemochromatosis (HH) is the most common autosomal recessive disorder known in humans. A candidate gene for HH called HFE has recently been cloned that encodes a novel member of the major histocompatibility complex class I family. Most HH patients are homozygous for a Cys-282→Tyr (C282Y) mutation in HFE gene, which has been shown to disrupt interaction with β2-microglobulin; a second mutation, His-63→Asp (H63D), is enriched in HH patients who are heterozygous for C282Y mutation. The aims of this study were to determine the effects of the C282Y and H63D mutations on the cellular trafficking and degradation of the HFE protein in transfected COS-7 cells. The results indicate that, while the wild-type and H63D HFE proteins associate with β2-microglobulin and are expressed on the cell surface of COS-7 cells, these capabilities are lost by the C282Y HFE protein. We present biochemical and immunofluorescence data that indicate that the C282Y mutant protein: (i) is retained in the endoplasmic reticulum and middle Golgi compartment, (ii) fails to undergo late Golgi processing, and (iii) is subject to accelerated degradation. The block in intracellular transport, accelerated turnover, and failure of the C282Y protein to be presented normally on the cell surface provide a possible basis for impaired function of this mutant protein in HH.

L-arginine-dependent nitric oxide production of a murine macrophage-like RAW 264.7 cell line stimulated with Porphyromonas gingivalis lipopolysaccharide

The aim of this study was to determine nitric oxide (NO) production of a murine macrophage cell line (RAW 264.7 cells) when stimulated with Porphyromonas gingivalis lipopolysaccharides (Pg-LPS). RAW264.7 cells were incubated with i) various concentrations of Pg-LPS or Salmonella typhosa LPS (St-LPS), ii) Pg-LPS with or without L-arginine and/or N-G-monomethyl-L-arginine (NMMA), an arginine analog or iii) Pg-LPS and interferon-gamma (IFN-gamma) with or without anti-IFN-gamma antibodies or interleukin-10 (IL-10). Tissue culture supernatants were assayed for NO levels after 24 h in culture. NO was not observed in tissue culture supernatants of RAW 264.7 cells following stimulation with Pg-LPS, but was observed after stimulation with St-LPS. Exogenous L-arginine restored the ability of Pg-LPS to induce NO production; however, the increase in NO levels of cells stimulated with Pg-LPS with exogenous L-arginine was abolished by NMMA. IFN-gamma induced independent NO production by Pg-LPS-stimulated macrophages and this stimulatory effect of IFN-gamma could be completely suppressed by anti-IFN-gamma antibodies and IL-10. These results suggest that Pg-LPS is able to stimulate NO production in the RAW264.7 macrophage cell model in an L-arginine-dependent mechanism which is itself independent of the action of IFN-gamma.

Antisense-mediated inhibition of the plasma membrane Calcium-ATPase suppresses proliferation of MCF-7 cells

Alterations in Ca2+ signaling may contribute to tumorigenesis and the mechanism of action of some anticancer drugs. The plasma membrane calcium-ATPase (PMCA) is a crucial controller of intracellular Ca2+ signaling. Altered PMCA expression occurs in the mammary gland during lactation and in breast cancer cell lines. Despite this, the consequences of PMCA inhibition in breast cancer cell lines have not been investigated. In this work, we used Tet-off PMCA antisense-expressing MCF-7 cells to assess the effects of PMCA inhibition in a human breast cancer cell line. At a level of PMCA inhibition that did not completely prevent PMCA-mediated Ca2+ efflux and did not induce cell death, a dramatic inhibition of cellular proliferation was observed. Fluorescence-activated cell sorting analysis indicated that PMCA antisense involves changes in cell cycle kinetics but not cell cycle arrest. We concluded that modulation of PMCA has important effects in regulating the proliferation of human breast cancer MCF-7 cells.

Peroxisome proliferator-activated receptor alpha expression is regulated by estrogen receptor alpha and modulates the response of MCF-7 cells to sodium butyrate

Peroxisome proliferator-activated receptors are ligand-activated transcription factors with a potential role in cancer. We investigated peroxisome proliferator-activated receptor alpha expression in breast cancer cell lines and showed a relationship between mean peroxisome proliferator-activated receptor alpha and estrogen receptor alpha mRNA levels in estrogen receptor alpha positive breast cancer cells. Transfection of estrogen receptor alpha into the estrogen receptor alpha negative cell line, MDA-MB-231 decreased peroxisome proliferator-activated receptor a mRNA and conversely inhibition of estrogen receptor alpha by ICI-182 780 in estrogen receptor a positive, MCF-7 cells increased peroxisome proliferator-activated receptor a mRNA levels. Estrogen receptor alpha levels can be modulated by histone deacetylase inhibitors and such agents are in clinical trials for cancer treatment. We found the histone deacetylase inhibitor, sodium butyrate, increased peroxisome proliferator-activated receptor alpha mRNA levels within 4 h of treatment. Peroxisome proliferator-activated receptor a modulation was independent of estrogen receptor alpha, as a similar increase was observed in the estrogen receptor a negative MDA-MB-231 cells. To further investigate the relationship between sodium butyrate and peroxisome proliferator-activated receptor alpha expression, we created an MCF-7 cell line that conditionally over-expresses human peroxisome proliferator-activated receptor alpha. Over-expression of the peroxisome proliferator-activated receptor protected MCF-7 cells from sodium butyrate-mediated inhibition of proliferation and attenuated sodium butyrate-mediated induction of histone deacetylase 3 mRNA, indicating that elevated levels of peroxisome proliferator-activated receptor alpha may reduce the sensitivity of cells to histone deacetylase inhibitors. The estrogen receptor alpha dependence of peroxisome proliferator-activated receptor alpha levels may be significant since estrogen receptor alpha negative breast cancer cells are associated with a more aggressive phenotype. Our studies also suggest that peroxisome proliferator-activated receptor alpha levels may be a marker of breast cancer cell sensitivity to histone deacetylase inhibitors. (c) 2004 Elsevier Ltd. All rights reserved.

Effects of propofol on lipopolysaccharide-induced expression and release of HMGB1 in macrophages

This study aimed to determine the effects of different concentrations of propofol (2,6-diisopropylphenol) on lipopolysaccharide (LPS)-induced expression and release of high-mobility group box 1 protein (HMGB1) in mouse macrophages. Mouse macrophage cell line RAW264.7 cells were randomly divided into 5 treatment groups. Expression levels of HMGB1 mRNA were detected using RT-PCR, and cell culture supernatant HMGB1 protein levels were detected using enzyme-linked immunosorbent assay (ELISA). Translocation of HMGB1 from the nucleus to the cytoplasm in macrophages was observed by Western blotting and activity of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the nucleus was detected using ELISA. HMGB1 mRNA expression levels increased significantly in the cell culture supernatant and in cells after 24 h of stimulating RAW264.7 cells with LPS (500 ng/mL). However, HMGB1 mRNA expression levels in the P2 and P3 groups, which received 500 ng/mL LPS with 25 or 50 μmol/mL propofol, respectively, were significantly lower than those in the group receiving LPS stimulation (P<0.05). After stimulation by LPS, HMGB1 protein levels were reduced significantly in the nucleus but were increased in the cytoplasm (P<0.05). Simultaneously, the activity of NF-κB was enhanced significantly (P<0.05). After propofol intervention, HMGB1 translocation from the nucleus to the cytoplasm and NF-κB activity were inhibited significantly (each P<0.05). Thus, propofol can inhibit the LPS-induced expression and release of HMGB1 by inhibiting HMGB1 translocation and NF-κB activity in RAW264.7 cells, suggesting propofol may be protective in patients with sepsis.

Rôle des récepteurs Toll-like et de la protéine adaptatrice MyD88 dans la régulation de l’hepcidine et le développement des hyposidérémies associées à l’inflammation

Le fer est un oligo-élément nécessaire pour le fonctionnement normal de toutes les cellules de l'organisme et joue un rôle essentiel dans de nombreuses fonctions biologiques. Cependant, le niveau de fer dans le corps doit être bien réglé, sinon la carence en fer entraine des divers états pathologiques tels que l'anémie et la diminution de l’immunité. D'autre part, une surcharge en fer potentialise la multiplication des germes, aggrave l’infection et la formation de radicaux libres ayant des effets toxiques sur les cellules et leurs composants, ce qui favorise les maladies cardio-vasculaires, l'inflammation et le cancer. L'hepcidine (HAMP), un régulateur négatif de l'absorption du fer, induit la dégradation de la ferroportine (FPN), le seul exportateur connu de fer ce qui réduit sa libération par les macrophages et inhibe son absorption gastro-intestinale. HAMP est synthétisé principalement par les hépatocytes, mais aussi par les macrophages. Cependant, il y a très peu de données sur la façon dont HAMP est régulé au niveau des macrophages. Plus récemment, nous avons constaté que l’induction de l’hepcidin dans le foie par le polysaccharide (LPS) est dépendante de la voie de signalisation médiée par « Toll-like receptor 4 » (TLR4). Grâce au TLR4, le LPS induit l'activation des macrophages qui sécrètent de nombreuses différentes cytokines inflammatoires, y compris Interleukine 6 (IL-6), responsable de l'expression de HAMP hépatique. Dans le premier chapitre de la présente étude, nous avons étudié la régulation de HAMP dans la lignée cellulaire macrophagique RAW264.7 et dans les macrophages péritonéaux murins stimulés par différents ligands des TLRs. Nous avons constaté que TLR2 et TLR4 par l'intermédiaire de la protéine adaptatrice « myeloid differentiation primary response gene 88 » (MyD88) activent l'expression de HAMP dans les cellules RAW264.7 et les macrophages péritonéaux sauvages murins, tandis que cette expression a été supprimée dans les macrophages isolés des souris TLR2-/-, TLR4-déficiente ou MyD88-/-. En outre, nous avons constaté que la production d'IL-6 par les cellules RAW264.7 stimulées avec du LPS a été renforcée par l’ajout des quantités élevées de fer dans le milieu de culture. Au cours de l’inflammation, le niveau de HAMP est fortement augmenté. Ainsi, lorsque l'inflammation persiste, l’expression de HAMP continue à être activée par des cytokines pro-inflammatoires conduisant à une hyposidérémie. Malgré que cette dernière soit considérée comme une défense de l'hôte pour priver les micro-organismes de fer, celle ci cause un développement d'anémies nommées anémies des maladies chroniques. Ainsi, dans le deuxième chapitre de la présente étude, nous avons étudié l'implication des TLRs et leurs protéines adaptatrices MyD88 et TIR-domain-containing adapter-inducing interferon-β (TRIF) dans le développement des hyposidérémies. En utilisant des souris déficientes en MyD88 et TRIF, nous avons montré que les voies de signalisations MyD88 et TRIF sont essentielles pour l’induction de HAMP par le LPS. Malgré l'absence de HAMP, les souris déficientes ont été capables de développer une hyposidérémie, mais la réponse des souris déficientes en MyD88 a été très légère, ce qui indique l'exigence de cette protéine pour assurer une réponse maximale au LPS. En outre, nous avons constaté que la signalisation MyD88 est nécessaire pour le stockage du fer au niveau de la rate, ainsi que l'induction de lipocaline 2 (LCN2), qui est une protéine impliquée dans la fixation du fer pour limiter la croissance bactérienne. Indépendamment de MyD88 ou TRIF, l'activation de TLR4 et TLR3 a conduit, au niveau de la rate, à une diminution rapide de l’expression de FPN et du « Human hemochromatosis protein » (HFE) qui est une protéine qui limite la séquestration du fer cellulaire à partir de la circulation. Cependant, malgré cette baisse d’expression, le manque de la signalisation MyD88 a altéré de manière significative la réponse hyposidérémique. En établissant le rôle des TLRs et de la protéine adaptatrice MyD88 dans la diminution du taux du fer sérique au cours de la réponse inflammatoire, nous avons remarqué qu’en réponse au surcharge en fer les souris déficientes en MyD88 accumulent de manière significative plus de fer hépatique par rapport aux souris sauvages, et cela indépendamment des TLRs. Ainsi, dans le troisième chapitre de la présente étude, nous avons étudié le phénotype observé chez les souris déficientes en MyD88. Nous avons trouvé que l'expression de HAMP chez ces souris a été plus faible que celle des souris de type sauvage. Pour cela, nous avons exploré la signalisation à travers la voie du « Bone Morphogenetic Proteins 6 » (BMP6) qui est considérée comme étant la voie fondamentale de la régulation de HAMP en réponse aux concentrations du fer intracellulaires et extracellulaires et nous avons trouvé que l'expression protéique de Smad4, un régulateur positif de l'expression de HAMP, est significativement plus faible chez les souris MyD88-/- par rapport aux souris sauvages. En outre, on a montré que MyD88 interagit avec « mothers against decapentaplegic, Drosophila, homolog 4 » (Smad4) et que cette interaction est essentielle pour l’induction de HAMP à travers la voie BMP6. En conclusion, notre étude montre que l'expression de HAMP dans les macrophages est régulée principalement par TLR2 et TLR4 à travers la voie MyD88 et que l'accumulation du fer dans les macrophages peut affecter les niveaux des cytokines pro-inflammatoires. En outre, nos analyses démontrent que le développement d’hyposidérémie en réponse au LPS se produit par l'intermédiaire d’un mécanisme dépendant de MyD88 qui est dissociée de la production de cytokines et de HAMP. En plus, nos recherches montrent que MyD88 est nécessaire pour l'expression de Smad4 et cela pour garantir une réponse optimale à travers la signalisation BMP6, conduisant ainsi à une expression adéquate de HAMP. Enfin, la protéine MyD88 joue un rôle crucial dans, la régulation de HAMP au niveau des macrophages, la diminution du taux du fer sérique en réponse au LPS et le maintien de l'homéostasie du fer.

Efecto de la hipoxia-reoxigenación y las radiaciones ionizantes en la captación de glucosa en líneas tumorales de seno y colon cocultivadas con células endoteliales.

La captación de glucosa y su conversión en lactato juega un papel fundamental en el metabolismo tumoral, independientemente de la concentración de oxígeno presente en el tejido (efecto Warburg). Sin embrago, dicha captación varía de un tipo tumoral a otro, y dentro del mismo tumor, situación que podría depender de las características microambientales tumorales (fluctuaciones de oxígeno, presencia de otros tipos celulares) y de factores estresores asociados a los tratamientos. Se estudió el efecto de la hipoxia-reoxigenación (HR) y las radiaciones ionizantes (RI) sobre la captación de glucosa, en cultivos de líneas tumorales MCF-7 y HT-29, cultivadas de forma aislada o en cocultivo con la línea celular EAhy296. Se encontró que la captación de glucosa en HR es diferente para lo descrito en condiciones de hipoxia permanente y que es modificada en el cocultivo. Se identificaron poblaciones celulares dentro de la misma línea celular, de alta y baja captación de glucosa, lo que implicaría una simbiosis metabólica de la célula como respuesta adaptativa a las condiciones tumorales. Se evaluó la expresión de NRF2 y la translocación nuclear de NRF2 y HIF1a, como vías de respuesta a estrés celular e hipoxia. La translocación nuclear de las proteínas evaluadas explicaría el comportamiento metabólico de las células tumorales de seno, pero no de colon, por lo cual deben existir otras vías metabólicas implicadas. Las diferencias en el comportamiento de las células tumorales en HR en relación con hipoxia permitirá realizar planeaciones dosimétricas más dinámicas, que reevalúen las condiciones de oxigenación tumoral constantemente.

11-Oxoaerothionin isolated from the marine sponge Aplysina fistularis shows anti-inflammatory activity in LPS-stimulated macrophages

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)