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1.
Clin Sci (Lond) ; 138(10): 573-597, 2024 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-38718356

RESUMO

The three striatins (STRN, STRN3, STRN4) form the core of STRiatin-Interacting Phosphatase and Kinase (STRIPAK) complexes. These place protein phosphatase 2A (PP2A) in proximity to protein kinases thereby restraining kinase activity and regulating key cellular processes. Our aim was to establish if striatins play a significant role in cardiac remodelling associated with cardiac hypertrophy and heart failure. All striatins were expressed in control human hearts, with up-regulation of STRN and STRN3 in failing hearts. We used mice with global heterozygote gene deletion to assess the roles of STRN and STRN3 in cardiac remodelling induced by angiotensin II (AngII; 7 days). Using echocardiography, we detected no differences in baseline cardiac function or dimensions in STRN+/- or STRN3+/- male mice (8 weeks) compared with wild-type littermates. Heterozygous gene deletion did not affect cardiac function in mice treated with AngII, but the increase in left ventricle mass induced by AngII was inhibited in STRN+/- (but not STRN3+/-) mice. Histological staining indicated that cardiomyocyte hypertrophy was inhibited. To assess the role of STRN in cardiomyocytes, we converted the STRN knockout line for inducible cardiomyocyte-specific gene deletion. There was no effect of cardiomyocyte STRN knockout on cardiac function or dimensions, but the increase in left ventricle mass induced by AngII was inhibited. This resulted from inhibition of cardiomyocyte hypertrophy and cardiac fibrosis. The data indicate that cardiomyocyte striatin is required for early remodelling of the heart by AngII and identify the striatin-based STRIPAK system as a signalling paradigm in the development of pathological cardiac hypertrophy.


Assuntos
Angiotensina II , Cardiomegalia , Camundongos Knockout , Miócitos Cardíacos , Animais , Angiotensina II/farmacologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Cardiomegalia/genética , Cardiomegalia/patologia , Cardiomegalia/metabolismo , Cardiomegalia/fisiopatologia , Masculino , Humanos , Proteínas Musculares/metabolismo , Proteínas Musculares/genética , Remodelação Ventricular , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Ligação a Calmodulina , Proteínas do Tecido Nervoso
2.
Proc Biol Sci ; 287(1940): 20202523, 2020 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-33259764

RESUMO

An important question in cancer evolution concerns which traits make a cell likely to successfully metastasize. Cell motility phenotypes, mediated by cell shape change, are strong candidates. We experimentally evolved breast cancer cells in vitro for metastatic capability, using selective regimes designed to simulate stages of metastasis, then quantified their motility behaviours using computer vision. All evolved lines showed changes to motility phenotypes, and we have identified a previously unknown density-dependent motility phenotype only seen in cells selected for colonization of decellularized lung tissue. These cells increase their rate of morphological change with an increase in migration speed when local cell density is high. However, when the local cell density is low, we find the opposite relationship: the rate of morphological change decreases with an increase in migration speed. Neither the ancestral population, nor cells selected for their ability to escape or invade extracellular matrix-like environments, displays this dynamic behavioural switch. Our results suggest that cells capable of distant-site colonization may be characterized by dynamic morphological phenotypes and the capacity to respond to the local social environment.


Assuntos
Neoplasias da Mama , Movimento Celular , Fenótipo , Animais , Feminino , Humanos , Pulmão
3.
Exp Cell Res ; 370(2): 417-425, 2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-30003879

RESUMO

Focal adhesions (FAs) play an important role in cancer cell migration and metastasis by linking the actin cytoskeleton to the extracellular matrix, allowing the cell to generate traction. SUMOylation is a post-translational modification of proteins on lysine residues that can affect protein localisation, turnover and protein-protein interactions. In this study, we demonstrate that talin, a key component of FAs, can be post-translationally modified by SUMOylation in MDA-MB-231 breast cancer cells and U2OS osteosarcoma cells. Furthermore we demonstrate that SUMOylation regulates the dynamic activities of FAs including their number, size and turnover rate. Inhibiting SUMOylation significantly reduced the speed of cell migration. The identification of talin as a SUMO target provides insight into the mechanisms regulating focal adhesion formation and turnover and potentially identifies a novel mechanism underlying cell migration.


Assuntos
Movimento Celular/fisiologia , Adesões Focais/metabolismo , Sumoilação/fisiologia , Talina/metabolismo , Citoesqueleto de Actina/metabolismo , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Matriz Extracelular/metabolismo , Humanos , Vinculina/metabolismo
4.
J Appl Toxicol ; 34(9): 1051-9, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24652746

RESUMO

Alkyl esters of p-hydroxybenzoic acid (parabens) are widely used as preservatives in personal care products, foods and pharmaceuticals. Their oestrogenic activity, their measurement in human breast tissue and their ability to drive proliferation of oestrogen-responsive human breast cancer cells has opened a debate on their potential to influence breast cancer development. As proliferation is not the only hallmark of cancer cells, we have investigated the effects of exposure to parabens at concentrations of maximal proliferative response on migratory and invasive properties using three oestrogen-responsive human breast cancer cell lines (MCF-7, T-47-D, ZR-75-1). Cells were maintained short-term (1 week) or long-term (20 ± 2 weeks) in phenol-red-free medium containing 5% charcoal-stripped serum with no addition, 10(-8) M 17ß-oestradiol, 1-5 × 10(-4) M methylparaben, 10(-5) M n-propylparaben or 10(-5) M n-butylparaben. Long-term exposure (20 ± 2 weeks) of MCF-7 cells to methylparaben, n-propylparaben or n-butylparaben increased migration as measured using a scratch assay, time-lapse microscopy and xCELLigence technology: invasive properties were found to increase in matrix degradation assays and migration through matrigel on xCELLigence. Western immunoblotting showed an associated downregulation of E-cadherin and ß-catenin in the long-term paraben-exposed cells which could be consistent with a mechanism involving epithelial to mesenchymal transition. Increased migratory activity was demonstrated also in long-term paraben-exposed T-47-D and ZR-75-1 cells using a scratch assay and time-lapse microscopy. This is the first report that in vitro, parabens can influence not only proliferation but also migratory and invasive properties of human breast cancer cells.


Assuntos
Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Parabenos/toxicidade , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Regulação para Baixo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Estradiol/metabolismo , Estrogênios/metabolismo , Feminino , Humanos , Células MCF-7 , Invasividade Neoplásica , Conservantes Farmacêuticos/toxicidade , beta Catenina/genética , beta Catenina/metabolismo
5.
Am J Physiol Heart Circ Physiol ; 304(10): H1302-13, 2013 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-23504181

RESUMO

Prolonged hemodynamic load as a result of hypertension eventually leads to maladaptive cardiac adaptation and heart failure. The signaling pathways that underlie these changes are still poorly understood. The adaptive response to mechanical load is mediated by mechanosensors that convert the mechanical stimuli into a biological response. We examined the effect of cyclic mechanical stretch on myocyte adaptation using neonatal rat ventricular myocytes with 10% (adaptive) or 20% (maladaptive) maximum strain at 1 Hz for 48 h to mimic in vivo mechanical stress. Cells were also treated with and without nitro-L-arginine methyl ester (L-NAME), a general nitric oxide synthase (NOS) inhibitor to suppress NO production. Maladaptive 20% mechanical stretch led to a significant loss of intact sarcomeres that were rescued by L-NAME (P < 0.05; n ≥ 5 cultures). We hypothesized that the mechanism was through NO-induced alteration of myocyte gene expression. L-NAME upregulated the mechanosensing proteins muscle LIM protein (MLP; by 100%; P < 0.05; n = 5 cultures) and lipoma preferred partner (LPP), a novel cardiac protein (by 80%; P < 0.05; n = 4 cultures). L-NAME also significantly altered the subcellular localization of LPP and MLP in a manner that favored growth and adaptation. These findings suggest that NO participates in stretch-mediated adaptation. The use of isoform selective NOS inhibitors indicated a complex interaction between inducible NOS and neuronal NOS isoforms regulate gene expression. LPP knockdown by small intefering RNA led to formation of α-actinin aggregates and Z bodies showing that myofibrillogenesis was impaired. There was an upregulation of E3 ubiquitin ligase (MUL1) by 75% (P < 0.05; n = 5 cultures). This indicates that NO contributes to stretch-mediated adaptation via the upregulation of proteins associated with mechansensing and myofibrillogenesis, thereby presenting potential therapeutic targets during the progression of heart failure.


Assuntos
Expressão Gênica/fisiologia , Proteínas com Domínio LIM/fisiologia , Proteínas dos Microfilamentos/fisiologia , Desenvolvimento Muscular/fisiologia , Miócitos Cardíacos/fisiologia , Óxido Nítrico/fisiologia , Proteínas Oncogênicas/fisiologia , Actinina/metabolismo , Actinina/fisiologia , Animais , Western Blotting , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Proteínas dos Microfilamentos/genética , Desenvolvimento Muscular/efeitos dos fármacos , Desenvolvimento Muscular/genética , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Proteínas Oncogênicas/genética , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Sarcômeros/fisiologia , Frações Subcelulares/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/fisiologia
6.
Stem Cells ; 30(6): 1182-95, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22593017

RESUMO

Skeletal muscle undergoes a progressive age-related loss in mass and function. Preservation of muscle mass depends in part on satellite cells, the resident stem cells of skeletal muscle. Reduced satellite cell function may contribute to the age-associated decrease in muscle mass. Here, we focused on characterizing the effect of age on satellite cell migration. We report that aged satellite cells migrate at less than half the speed of young cells. In addition, aged cells show abnormal membrane extension and retraction characteristics required for amoeboid-based cell migration. Aged satellite cells displayed low levels of integrin expression. By deploying a mathematical model approach to investigate mechanism of migration, we have found that young satellite cells move in a random "memoryless" manner, whereas old cells demonstrate superdiffusive tendencies. Most importantly, we show that nitric oxide, a key regulator of cell migration, reversed the loss in migration speed and reinstated the unbiased mechanism of movement in aged satellite cells. Finally, we found that although hepatocyte growth factor increased the rate of aged satellite cell movement, it did not restore the memoryless migration characteristics displayed in young cells. Our study shows that satellite cell migration, a key component of skeletal muscle regeneration, is compromised during aging. However, we propose clinically approved drugs could be used to overcome these detrimental changes.


Assuntos
Células-Tronco Adultas/citologia , Envelhecimento/fisiologia , Movimento Celular/fisiologia , Músculo Esquelético/citologia , Fatores Etários , Animais , Processos de Crescimento Celular/fisiologia , Embrião de Galinha , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regeneração/fisiologia
7.
Cell Signal ; 81: 109934, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33545231

RESUMO

BACKGROUND: Lung cancer has a poor prognosis partly due to a lack of response to treatments such as the chemotherapy drug gemcitabine. Combinations of chemotherapy drugs with signal transduction inhibitors may be more effective treatments. In this study we have investigated the impact of targeting the mTOR signalling pathway on the efficacy of gemcitabine in different cancer cell lines. METHODS: Time-lapse microscopy, immuno-staining, and western blot techniques were used to evaluate the efficacy of applied treatments either in measuring phosphorylation levels of mTOR down-stream targets or in tracking down the fate of targeted cells. Reactive oxygen species and relative levels of protein phosphorylation were also quantified. For comparison between treated groups t-test and analysis of variance test were applied. RESULTS: Our data showed that mTORC1 has no role in sensitising A549 lung cancer cells to gemcitabine. However, targeting mTORC1/2 with the pharmacological inhibitor torin1 or by over-expressing Deptor, the negative regulator of mTOR signalling, sensitised these cells to gemcitabine. Silencing mTORC2, but not mTORC1, induced apoptosis and significantly improved the apoptosis-inducing effects of gemcitabine. Results also suggest that Rictor is required to maintain cell survival through modulating p38α, ERK1/2, RSK1/2/3 and the transcription factor STAT3. Multiple cell line comparisons revealed that PANC-1 pancreatic cancer cells were also sensitive to mTOR inhibition, but MCF7 breast cancer, MCF10A breast epithelial and H727 lung cancer cell lines were more resistant to the treatment. CONCLUSIONS: Inhibition of mTORC2 may have benefits in the treatment of gemcitabine resistant cancers, and the genetic background of the cell line may determine its response to mTOR inhibition.


Assuntos
Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Pulmonares/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proteínas de Neoplasias/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Células A549 , Desoxicitidina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Sistema de Sinalização das MAP Quinases/genética , Células MCF-7 , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Proteínas de Neoplasias/genética , Serina-Treonina Quinases TOR/genética , Gencitabina
8.
R Soc Open Sci ; 7(4): 191645, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32431868

RESUMO

Tumour evolution depends on heritable differences between cells in traits affecting cell survival or replication. It is well established that cancer cells are genetically and phenotypically heterogeneous; however, the extent to which this phenotypic variation is heritable is far less well explored. Here, we estimate the broad-sense heritability (H 2) of two cell traits related to cancer hallmarks--cell motility and generation time--within populations of four cancer cell lines in vitro and find that motility is strongly heritable. This heritability is stable across multiple cell generations, with heritability values at the high end of those measured for a range of traits in natural populations of animals or plants. These findings confirm a central assumption of cancer evolution, provide a first quantification of the evolvability of key traits in cancer cells and indicate that there is ample raw material for experimental evolution in cancer cell lines. Generation time, a trait directly affecting cell fitness, shows substantially lower values of heritability than cell speed, consistent with its having been under directional selection removing heritable variation.

9.
Essays Biochem ; 63(5): 631-637, 2019 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-31654055

RESUMO

The complexity of biological systems creates challenges for fully understanding their behaviour. This is particularly true for cell migration which requires the co-ordinated activity of hundreds of individual components within cells. Mathematical modelling can help understand these complex systems by breaking the system into discrete steps which can then be interrogated in silico. In this review, we highlight scenarios in cell migration where mathematical modelling can be applied and discuss what types of modelling are most suited. Almost any aspect of cell migration is amenable to mathematical modelling from the modelling of intracellular processes such as chemokine receptor signalling and actin filament branching to larger scale processes such as the movement of individual cells or populations of cells through their environment. Two common ways of approaching this modelling are the use of models based on differential equations or agent-based modelling. The application of both these approaches to cell migration are discussed with specific examples along with common software tools to facilitate the process for non-mathematicians. We also highlight the challenges of modelling cell migration and the need for rigorous experimental work to effectively parameterise a model.


Assuntos
Movimento Celular , Modelos Biológicos , Simulação por Computador , Software , Análise de Sistemas
10.
Eur J Pharmacol ; 862: 172627, 2019 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-31461638

RESUMO

Isorhapontigenin is a polyphenolic compound found in Chinese herbs and grapes. It is a methoxylated analogue of a stilbenoid, resveratrol, which is well-known for its various beneficial effects including anti-platelet activity. Isorhapontigenin possesses greater oral bioavailability than resveratrol and has also been identified to possess anti-cancer and anti-inflammatory properties. However, its effects on platelet function have not been reported previously. In this study, we report the effects of isorhapontigenin on the modulation of platelet function. Isorhapontigenin was found to selectively inhibit ADP-induced platelet aggregation with an IC50 of 1.85 µM although it displayed marginal inhibition on platelet aggregation induced by other platelet agonists at 100 µM. However, resveratrol exhibited weaker inhibition on ADP-induced platelet aggregation (IC50 > 100 µM) but inhibited collagen induced platelet aggregation at 50 µM and 100 µM. Isorhapontigenin also inhibited integrin αIIbß3 mediated inside-out and outside-in signalling and dense granule secretion in ADP-induced platelet activation but interestingly, no effect was observed on α-granule secretion. Isorhapontigenin did not exert any cytotoxicity on platelets at the concentrations of up to 100 µM. Furthermore, it did not affect haemostasis in mice at the IC50 concentration (1.85 µM). In addition, the mechanistic studies demonstrated that isorhapontigenin increased cAMP levels and VASP phosphorylation at Ser157 and decreased Akt phosphorylation. This suggests that isorhapontigenin may interfere with cAMP and PI3K signalling pathways that are associated with the P2Y12 receptor. Molecular docking studies emphasised that isorhapontigenin has greater binding affinity to P2Y12 receptor than resveratrol. Our results demonstrate that isorhapontigenin has selective inhibitory effects on ADP-stimulated platelet activation possibly via P2Y12 receptor.


Assuntos
Plaquetas/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Estilbenos/farmacologia , Difosfato de Adenosina/farmacologia , Animais , Plaquetas/metabolismo , Avaliação Pré-Clínica de Medicamentos , Feminino , Voluntários Saudáveis , Humanos , Concentração Inibidora 50 , Masculino , Camundongos , Modelos Animais , Simulação de Acoplamento Molecular , Inibidores da Agregação Plaquetária/uso terapêutico , Testes de Função Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Receptores Purinérgicos P2Y12/química , Receptores Purinérgicos P2Y12/metabolismo , Resveratrol/análogos & derivados , Resveratrol/farmacologia , Transdução de Sinais/efeitos dos fármacos , Estilbenos/química , Estilbenos/uso terapêutico , Trombose/tratamento farmacológico
11.
Hum Reprod ; 23(8): 1733-41, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18487214

RESUMO

BACKGROUND: Trophoblast invasion is a temporally and spatially regulated scheme of events that can dictate pregnancy outcome. Evidence suggests that the potent mitogen epidermal growth factor (EGF) regulates cytotrophoblast (CTB) differentiation and invasion during early pregnancy. METHODS AND RESULTS: In the present study, the first trimester extravillous CTB cell line SGHPL-4 was used to investigate the signalling pathways involved in the motile component of EGF-mediated CTB migration/invasion. EGF induced the phosphorylation of the phosphatidylinositol 3-kinase (PI3-K)-dependent proteins, Akt and GSK-3beta as well as both p42/44 MAPK and p38 mitogen-activated protein kinases (MAPK). EGF-stimulated motility was significantly reduced following the inhibition of PI3-K (P < 0.001), Akt (P < 0.01) and both p42/44 MAPK (P < 0.001) and p38 MAPKs (P < 0.001) but not the inhibition of GSK-3beta. Further analysis indicated that the p38 MAPK inhibitor SB 203580 inhibited EGF-stimulated phosphorylation of Akt on serine 473, which may be responsible for the effect SB 203580 has on CTB motility. Although Akt activation leads to GSK-3beta phosphorylation and the subsequent expression of beta-catenin, activation of this pathway by 1-azakenpaullone was insufficient to stimulate the motile phenotype. CONCLUSION: We demonstrate a role for PI3-K, p42/44 MAPK and p38 MAPK in the stimulation of CTB cell motility by EGF, however activation of beta-catenin alone was insufficient to stimulate cell motility.


Assuntos
Movimento Celular/fisiologia , Fator de Crescimento Epidérmico/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Trofoblastos/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Benzazepinas/farmacologia , Movimento Celular/efeitos dos fármacos , Cromonas/farmacologia , Feminino , Flavonoides/farmacologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta , Humanos , Indóis/farmacologia , Morfolinas/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Gravidez , Primeiro Trimestre da Gravidez , beta Catenina/metabolismo
12.
Metabolism ; 56(7): 865-75, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17570244

RESUMO

We hypothesized that the hepatotoxicity that develops after the induction of oxidative stress (induced by d-galactosamine [GalN]) can be ameliorated by alpha-tocopherol (ATC) and the soy isoflavone daidzein. To test this, we ranked and assigned male Wistar rats into 6 groups, which involved pretreatment (ATC or daidzein) for 1 hour followed by treatment (GalN) for 23 hours. Histopathologic analysis showed that GalN administration induced marked necrosis (P < .001), steatosis (P < .001), both lobular and portal inflammations (P < .001), overall histopathologic score (P < .001), and activation of caspase-3 in the liver (P < .001). Immunohistochemical staining of malondialdehyde-protein adducts, a measure of oxidative stress, was increased in response to GalN (P < .001). Paradoxically, there were increases in total (P < .05) and cytosolic superoxide dismutase (P < .001) activities after GalN administration, indicative of an up-regulation of antioxidant defenses. The concentration of total protein (P < .001), albumin (P < .01), and globulin fractions (P < .001) in the plasma, as well as the activity of aspartate aminotransferase (P < .001), was significantly perturbed after GalN treatment, reflective of overall acute hepatic injury. Administration of daidzein showed a significant amelioration of the Ga1N-induced increase in malondialdehyde-protein adducts (P < .01) and cytosolic superoxide dismutase activities (P < .01) in the liver. However, all other variables were not significantly altered in response to daidzein. In response to ATC pretreatment, the total histopathologic score (P < .05), degree of necrosis (P < .05), and both lobular (P < .05) and portal (P = .05) inflammations were significantly ameliorated. To conclude, both daidzein and ATC protect the liver against oxidative damage possibly via different pathways.


Assuntos
Citoproteção , Galactosamina/toxicidade , Isoflavonas/farmacologia , Fígado/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , alfa-Tocoferol/farmacologia , Animais , Glutationa Peroxidase/metabolismo , Fígado/metabolismo , Fígado/patologia , Masculino , Malondialdeído/metabolismo , Ratos , Ratos Wistar , Superóxido Dismutase/metabolismo
13.
Cell Signal ; 17(5): 571-80, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15683732

RESUMO

Excessive apoptosis of trophoblast cells is thought to be a contributing factor in complications of pregnancy such as pre-eclampsia. Hepatocyte growth factor (HGF) inhibits apoptosis in trophoblasts and we have investigated the signalling pathways through which this anti-apoptotic effect is mediated. Treatment of cells with HGF led to rapid phosphorylation of Akt while an Akt inhibitor blocked the protective effect of HGF. Glycogen synthase kinase-3beta (GSK-3beta) was found to be one of the downstream targets of Akt. HGF treatment inactivated GSK-3beta which in turn led to the activation of the transcription factor beta-catenin. Pharmacological inhibition of GSK-3beta, independently of HGF treatment, strongly increased both beta-catenin activity and cell survival, suggesting that beta-catenin alone has a pronounced anti-apoptotic effect. We also found that both HGF treatment and pharmacological activation of beta-catenin leads to increased expression of inducible nitric oxide synthase (iNOS). We suggest that the Akt mediated activation of beta-catenin leads to inhibition of trophoblast apoptosis following increased expression of iNOS.


Assuntos
Apoptose , Proteínas do Citoesqueleto/metabolismo , Fator de Crescimento de Hepatócito/farmacologia , Óxido Nítrico Sintase/biossíntese , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Trofoblastos/metabolismo , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Proteínas I-kappa B/metabolismo , Inibidor de NF-kappaB alfa , NF-kappa B/metabolismo , Óxido Nítrico Sintase/fisiologia , Óxido Nítrico Sintase Tipo II , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Trofoblastos/citologia , Trofoblastos/enzimologia , Regulação para Cima , beta Catenina
14.
Arterioscler Thromb Vasc Biol ; 25(1): 102-8, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15499040

RESUMO

OBJECTIVE: Invasion of uterine spiral arteries by extravillous trophoblasts in the first trimester of pregnancy results in loss of endothelial and musculoelastic layers. This remodeling is crucial for an adequate blood supply to the fetus with a failure to remodel implicated in the etiology of the hypertensive disorder preeclampsia. The mechanism by which trophoblasts induce this key process is unknown. This study gives the first insights into the potential mechanisms involved. METHODS AND RESULTS: Spiral arteries were dissected from nonplacental bed biopsies obtained at Caesarean section, and a novel model was used to mimic in vivo events. Arteries were cultured with trophoblasts in the lumen, and apoptotic changes in the endothelial layer were detected after 20 hours, leading to loss of endothelium by 96 hours. In vitro, coculture experiments showed that trophoblasts stimulated apoptosis of primary decidual endothelial cells and an endothelial cell line. This was blocked by caspase inhibition and NOK2, a FasL blocking antibody. NOK2 also abrogated trophoblast-induced endothelial apoptosis in the vessel model. CONCLUSIONS: Extravillous trophoblast induction of endothelial apoptosis is a possible mechanism by which the endothelium is removed, and vascular remodeling may occur in uterine spiral arteries. Fas/FasL interactions have an important role in trophoblast-induced endothelial apoptosis.


Assuntos
Apoptose/fisiologia , Artérias/metabolismo , Células Endoteliais/patologia , Glicoproteínas de Membrana/metabolismo , Trofoblastos/metabolismo , Útero/irrigação sanguínea , Receptor fas/metabolismo , Artérias/patologia , Células Cultivadas , Endotélio Vascular/patologia , Proteína Ligante Fas , Feminino , Humanos
15.
J Gerontol A Biol Sci Med Sci ; 69(9): 1049-59, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24414825

RESUMO

Mammalian aging is accompanied by a progressive loss of skeletal muscle, a process called sarcopenia. Myostatin, a secreted member of the transforming growth factor-ß family of signaling molecules, has been shown to be a potent inhibitor of muscle growth. Here, we examined whether muscle growth could be promoted in aged animals by antagonizing the activity of myostatin through the neutralizing activity of the myostatin propeptide. We show that a single injection of an AAV8 virus expressing the myostatin propeptide induced an increase in whole body weights and all muscles examined within 7 weeks of treatment. Our cellular studies demonstrate that muscle enlargement was due to selective fiber type hypertrophy, which was accompanied by a shift toward a glycolytic phenotype. Our molecular investigations elucidate the mechanism underpinning muscle hypertrophy by showing a decrease in the expression of key genes that control ubiquitin-mediated protein breakdown. Most importantly, we show that the hypertrophic muscle that develops as a consequence of myostatin propeptide in aged mice has normal contractile properties. We suggest that attenuating myostatin signaling could be a very attractive strategy to halt and possibly reverse age-related muscle loss.


Assuntos
Envelhecimento/fisiologia , Miostatina/antagonistas & inibidores , Peptídeos/farmacologia , Animais , Peso Corporal , Vetores Genéticos , Hipertrofia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Musculares/metabolismo , Músculo Esquelético/anatomia & histologia , Músculo Esquelético/patologia , Músculo Esquelético/fisiologia , Miostatina/fisiologia , Reação em Cadeia da Polimerase , Transdução de Sinais/fisiologia
16.
Evol Appl ; 6(3): 535-48, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23745144

RESUMO

Evolutionary processes play a central role in the development, progression and response to treatment of cancers. The current challenge facing researchers is to harness evolutionary theory to further our understanding of the clinical progression of cancers. Central to this endeavour will be the development of experimental systems and approaches by which theories of cancer evolution can be effectively tested. We argue here that the experimental evolution approach - whereby evolution is observed in real time and which has typically employed microorganisms - can be usefully applied to cancer. This approach allows us to disentangle the ecological causes of natural selection, identify the genetic basis of evolutionary changes and determine their repeatability. Cell cultures used in cancer research share many of the desirable traits that make microorganisms ideal for studying evolution. As such, experimental cancer evolution is feasible and likely to give great insight into the selective pressures driving the evolution of clinically destructive cancer traits. We highlight three areas of evolutionary theory with importance to cancer biology that are amenable to experimental evolution: drug resistance, social evolution and resource competition. Understanding the diversity, persistence and evolution of cancers is vital for treatment and drug development, and an experimental evolution approach could provide strategic directions and focus for future research.

17.
Eur J Cell Biol ; 91(3): 171-9, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22284833

RESUMO

Throughout pregnancy the cytotrophoblast, the stem cell of the placenta, gives rise to the differentiated forms of trophoblasts. The two main cell lineages are the syncytiotrophoblast and the invading extravillous trophoblast. A successful pregnancy requires extravillous trophoblasts to migrate and invade through the decidua and then remodel the maternal spiral arteries. Many invasive cells use specialised cellular structures called invadopodia or podosomes in order to degrade extracellular matrix. Despite being highly invasive cells, the presence of invadapodia or podosomes has not previously been investigated in trophoblasts. In this study these structures have been identified and characterised in extravillous trophoblasts. The role of specialised invasive structures in trophoblasts in the degradation of the extracellular matrix was compared with well characterised podosomes and invadopodia in other invasive cells and the trophoblast specific structures were characterised by using a sensitive matrix degradation assay which enabled visualisation of the structures and their dynamics. We show trophoblasts form actin rich protrusive structures which have the ability to degrade the extracellular matrix during invasion. The degradation ability and dynamics of the structures closely resemble podosomes, but have unique characteristics that have not previously been described in other cell types. The composition of these structures does not conform to the classic podosome structure, with no distinct ring of plaque proteins such as paxillin or vinculin. In addition, trophoblast podosomes protrude more deeply into the extracellular matrix than established podosomes, resembling invadopodia in this regard. We also show several significant pathways such as Src kinase, MAPK kinase and PKC along with MMP-2 and 9 as key regulators of extracellular matrix degradation activity in trophoblasts, while podosome activity was regulated by the rigidity of the extracellular matrix.


Assuntos
Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestrutura , Trofoblastos/metabolismo , Trofoblastos/ultraestrutura , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Actinas/metabolismo , Actinas/ultraestrutura , Linhagem Celular Tumoral , Decídua/metabolismo , Decídua/ultraestrutura , Feminino , Humanos , Pré-Eclâmpsia/metabolismo , Pré-Eclâmpsia/patologia , Gravidez
18.
FEBS Open Bio ; 2: 135-44, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23650592

RESUMO

Adaptor proteins play an important role in signaling pathways by providing a platform on which many other proteins can interact. Malfunction or mislocalization of these proteins may play a role in the development of disease. Lipoma preferred partner (LPP) is a nucleocytoplasmic shuttling adaptor protein. Previous work shows that LPP plays a role in the function of smooth muscle cells and in atherosclerosis. In this study we wanted to determine whether LPP has a role in the myocardium. LPP expression increased by 56% in hearts from pressure overload aortic-banded rats (p < 0.05 n = 4), but not after myocardial infarction, suggesting hemodynamic load regulates its expression. In vitro, LPP expression was 87% higher in cardiac fibroblasts than myocytes (p < 0.05 n = 3). LPP expression was downregulated in the absence of the actin cytoskeleton but not when microtubules were disassembled. We mechanically stretched cardiac fibroblasts using the Flexcell 4000 for 48 h (1 Hz, 5% maximum strain), which decreased total LPP total expression and membrane localization in subcellular fractions (p < 0.05, n = 5). However, L-NAME, an inhibitor of nitric oxide synthase (NOS), significantly upregulated LPP expression. These findings suggest that LPP is regulated by a complex interplay between NO and mechanical cues and may play a role in heart failure induced by increased hemodynamic load.

19.
Rejuvenation Res ; 14(3): 249-60, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21453013

RESUMO

Adult skeletal muscle possesses a resident stem cell population called satellite cells, which are responsible for tissue repair following damage. Satellite cell migration is crucial in promoting rapid tissue regeneration, but it is a poorly understood process. Furthermore, the mechanisms facilitating satellite cell movement have yet to be elucidated. This study investigates the process of satellite cell migration, revealing that they undergo two distinct phases of movement, first under the basal lamina and then rapidly increasing their velocity when on the myofiber surface. Most significantly, we show that satellite cells move using a highly dynamic blebbing or amoeboid-based mechanism and not via lamellipodia-mediated propulsion. We show that nitric oxide and noncanonical Wnt signaling pathways are necessary for regulating the formation of blebs and the migration of satellite cells. In summary, we propose that the formation of blebs and their necessity for satellite cell migration has significant implications in the future development of therapeutic regimes aimed at promoting skeletal muscle regeneration.


Assuntos
Células-Tronco Adultas/citologia , Envelhecimento/fisiologia , Movimento Celular , Extensões da Superfície Celular/metabolismo , Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/citologia , Actinas/metabolismo , Animais , Membrana Basal/metabolismo , Linhagem Celular , Polaridade Celular , Células Cultivadas , Citoesqueleto/metabolismo , Laminina/metabolismo , Camundongos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Células Satélites de Músculo Esquelético/enzimologia , Células Satélites de Músculo Esquelético/ultraestrutura , Transdução de Sinais , Proteínas Wnt/metabolismo , Quinases Associadas a rho/metabolismo
20.
Exp Cell Res ; 314(8): 1765-76, 2008 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-18394602

RESUMO

Nitric oxide regulates many important cellular processes including motility and invasion. Many of its effects are mediated through the modification of specific cysteine residues in target proteins, a process called S-nitrosylation. Here we show that S-nitrosylation of proteins occurs at the leading edge of migrating trophoblasts and can be attributed to the specific enrichment of inducible nitric oxide synthase (iNOS/NOS2) in this region. Localisation of iNOS to the leading edge is co-incidental with a site of extensive actin polymerisation and is only observed in actively migrating cells. In contrast endothelial nitric oxide synthase (eNOS/NOS3) shows distribution that is distinct and non-colocalised with iNOS, suggesting that the protein S-nitrosylation observed at the leading edge is caused only by iNOS and not eNOS. We have identified MMP-9 as a potential target for S-nitrosylation in these cells and demonstrate that it co-localises with iNOS at the leading edge of migrating cells. We further demonstrate that iNOS plays an important role in promoting trophoblast invasion, which is an essential process in the establishment of a successful pregnancy.


Assuntos
Movimento Celular , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico/metabolismo , Processamento de Proteína Pós-Traducional , Trofoblastos/enzimologia , Citoesqueleto de Actina/enzimologia , Linhagem Celular , Cisteína/metabolismo , Citoplasma/enzimologia , Humanos , Metaloproteinase 9 da Matriz/análise , Óxido Nítrico Sintase Tipo II/análise , Óxido Nítrico Sintase Tipo III/análise , Trofoblastos/fisiologia
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