RESUMO
The sodium-iodide symporter (NIS) is an integral membrane protein that plays a crucial role in iodide accumulation, especially in the thyroid. As for many other membrane proteins, its intracellular sorting and distribution have a tremendous effect on its function, and constitute an important aspect of its regulation. Many short sequences have been shown to contribute to protein trafficking along the sorting or endocytic pathways. Using bioinformatics tools, we identified such potential sites on human NIS [tyrosine-based motifs, SH2-(Src homology 2), SH3- and PDZ (post-synaptic density-95/discs large tumour suppressor/zonula occludens-1)-binding motifs, and diacidic, dibasic and dileucine motifs] and analysed their roles using mutagenesis. We found that several of these sites play a role in protein stability and/or targeting to the membrane. Aside from the mutation at position 178 (SH2 plus tyrosine-based motif) that affects iodide uptake, the most drastic effect is associated with the mutation of an internal PDZ-binding motif at position 121 that completely abolishes NIS expression at the plasma membrane. Mutating the sites located on the C-terminal domain of the protein has no effect except for the creation of a diacidic motif that decreases the total NIS protein level without affecting its expression at the plasma membrane.
Assuntos
Membrana Celular/metabolismo , Simportadores/metabolismo , Motivos de Aminoácidos , Membrana Celular/genética , Células HEK293 , Humanos , Domínios PDZ , Transporte Proteico/fisiologia , Simportadores/genética , Domínios de Homologia de srcRESUMO
Femur bone sections from a single donor were exposed for six months to (i) outdoor conditions (exposure to sun, rain, etc.); (ii) water-vapour saturated environment favourable to mould proliferation and (iii) humic-garden soil. Following these treatments, DNA was extracted and yields were compared with that of a control bone fragment kept under optimal laboratory storage conditions. Our results demonstrate that both mould and soil are very detrimental to bone DNA conservation since more than 97% of the bone DNA was lost in these samples as compared with the control condition. Outdoor exposure gives an intermediate result with 30% of the DNA still present in the bone. Thus, environments favourable to microorganisms proliferation appear detrimental to bone DNA conservation and are a bad prognostic should bone remains be used for genetic identification purpose. Comparatively, open-air exposure is much more favourable to bone DNA analysis.
Assuntos
DNA/isolamento & purificação , Fêmur/química , Manejo de Espécimes/métodos , Exposição Ambiental , Fêmur/patologia , Genética Forense , Patologia Legal , Fungos , Humanos , SoloRESUMO
Bone is the most challenging tissue for DNA extraction and purification. Expensive commercial kits and specific equipments are often used in forensic and anthropology laboratories towards that goal. We present here an integrated procedure that gives satisfactory results for DNA preparation from fresh, ancient or weathered bones. Extraction is performed under simple but efficient vacuum-controlled conditions that greatly limit the risks of cross-contaminations. The whole process has been designed to minimize the need for expensive equipment and chemicals, and to be compatible with any molecular biology laboratory. In addition, no toxic reagents are necessary and the procedure is straightforward. Combined with quantitative polymerase chain reaction (qPCR), this method allows species identification and sex determination from subcellular amount of DNA (1-5 pg). In addition, enough DNA is generally obtained for human DNA profiling if necessary. The whole procedure from bone treatment to the final qPCR results takes less than 48 hours. This procedure should allow any laboratory with standard molecular biology equipment and expertise to perform bone DNA characterization whenever necessary.
Assuntos
Osso e Ossos/química , DNA/isolamento & purificação , Manejo de Espécimes/métodos , Impressões Digitais de DNA , Humanos , Reação em Cadeia da Polimerase em Tempo Real , VácuoRESUMO
Fragile X syndrome, the most frequent form of inherited mental retardation, is due to the absence of Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein involved in several steps of RNA metabolism. To date, two RNA motifs have been found to mediate FMRP/RNA interaction, the G-quartet and the "kissing complex," which both induce translational repression in the presence of FMRP. We show here a new role for FMRP as a positive modulator of translation. FMRP specifically binds Superoxide Dismutase 1 (Sod1) mRNA with high affinity through a novel RNA motif, SoSLIP (Sod1 mRNA Stem Loops Interacting with FMRP), which is folded as three independent stem-loop structures. FMRP induces a structural modification of the SoSLIP motif upon its interaction with it. SoSLIP also behaves as a translational activator whose action is potentiated by the interaction with FMRP. The absence of FMRP results in decreased expression of Sod1. Because it has been observed that brain metabolism of FMR1 null mice is more sensitive to oxidative stress, we propose that the deregulation of Sod1 expression may be at the basis of several traits of the physiopathology of the Fragile X syndrome, such as anxiety, sleep troubles, and autism.
Assuntos
Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , Regulação da Expressão Gênica , RNA Mensageiro/metabolismo , Superóxido Dismutase/genética , Animais , Sítios de Ligação , Encéfalo/enzimologia , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Humanos , Camundongos , Camundongos Mutantes , Polirribossomos , Biossíntese de Proteínas , RNA Mensageiro/química , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1RESUMO
This interdisciplinary work focuses on the interest of a new auto-encoder for supervised classification of live cell populations growing in a thermostated imaging station and acquired by a Quantitative Phase Imaging (QPI) camera. This type of camera produces interferograms that have to be processed to extract features derived from quantitative linear retardance and birefringence measurements. QPI is performed on living populations without any manipulation or treatment of the cells. We use the efficient new autoencoder classification method instead of the classical Douglas-Rachford method. Using this new supervised autoencoder, we show that the accuracy of the classification of the cells present in the mitotic phase of the cell cycle is very high using QPI features. This is a very important finding since we demonstrate that it is now possible to very precisely follow cell growth in a non-invasive manner, without any bias. No dye or any kind of markers are necessary for this live monitoring. Any studies requiring analysis of cell growth or cellular response to any treatment could benefit from this new approach by simply monitoring the proportion of cells entering mitosis in the studied cell population.
Assuntos
Ciclo Celular/fisiologia , Processamento de Imagem Assistida por Computador/métodos , Índice Mitótico/métodos , Aprendizado de Máquina Supervisionado , Algoritmos , Ciclo Celular/efeitos dos fármacos , Células HeLa , Humanos , Microscopia , Nocodazol/farmacologiaRESUMO
We present here a label-free development based on preexisting Quantitative Phase Imaging (QPI) that allows non-invasive live monitoring of both individual cells and cell populations. Growth, death, effect of toxic compounds are quantified under visible light with a standard inverted microscope. We show that considering the global biomass of a cell population is a more robust and accurate method to assess its growth parameters in comparison to compiling individually segmented cells. This is especially true for confluent conditions. This method expands the use of light microscopy in answering biological questions concerning live cell populations even at high density. In contrast to labeling or lysis of cells this method does not alter the cells and could be useful in high-throughput screening and toxicity studies.
Assuntos
Microscopia/métodos , Biomassa , Linhagem Celular , Células HEK293 , Ensaios de Triagem em Larga Escala/métodos , HumanosRESUMO
BACKGROUND: Retroviral vectors are valuable tools for gene transfer. Particularly convenient are IRES-containing retroviral vectors expressing both the protein of interest and a marker protein from a single bicistronic mRNA. This coupled expression increases the relevance of tracking and/or selection of transduced cells based on the detection of a marker protein. pAP2 is a retroviral vector containing eGFP downstream of a modified IRES element of EMCV origin, and a CMV enhancer-promoter instead of the U3 region of the 5'LTR, which increases its efficiency in transient transfection. However, pAP2 contains a limited multicloning site (MCS) and shows weak eGFP expression, which previously led us to engineer an improved version, termed pPRIG, harboring: i) the wild-type ECMV IRES sequence, thereby restoring its full activity; ii) an optimized MCS flanked by T7 and SP6 sequences; and iii) a HA tag encoding sequence 5' of the MCS (pPRIG HAa/b/c). RESULTS: The convenience of pPRIG makes it a good basic vector to generate additional derivatives for an extended range of use. Here we present several novel pPRIG-based vectors (collectively referred to as PRIGs) in which : i) the HA tag sequence was inserted in the three reading frames 3' of the MCS (3'HA PRIGs); ii) a functional domain (ER, VP16 or KRAB) was inserted either 5' or 3' of the MCS (<< modular >> PRIGs); iii) eGFP was replaced by either eCFP, eYFP, mCherry or puro-R (<< single color/resistance >> PRIGs); and iv) mCherry, eYFP or eGFP was inserted 5' of the MCS of the IRES-eGFP, IRES-eCFP or IRES-Puro-R containing PRIGs, respectively (<< dual color/selection >> PRIGs). Additionally, some of these PRIGs were also constructed in a pMigR MSCV background which has been widely used in pluripotent cells. CONCLUSION: These novel vectors allow for straightforward detection of any expressed protein (3'HA PRIGs), for functional studies of chimeric proteins (<< modular >> PRIGs), for multiple transductions and fluorescence analyses of transduced cells (<< single color/resistance >> PRIGs), or for quantitative detection of studied proteins in independently identified/selected transduced cells (<< dual color/selection >> PRIGs). They maintain the original advantages of pPRIG and provide suitable tools for either transient or stable expression and functional studies in a large range of experimental settings.
Assuntos
Vírus da Encefalomiocardite/genética , Vetores Genéticos , Proteínas Luminescentes , Sequência de Bases , Clonagem Molecular/métodos , DNA Recombinante/síntese química , RNA Polimerases Dirigidas por DNA , Vírus da Encefalomiocardite/metabolismo , Elementos Facilitadores Genéticos , Genes Reporter , Vetores Genéticos/síntese química , Vetores Genéticos/metabolismo , Proteínas Luminescentes/síntese química , Proteínas Luminescentes/genética , Microscopia de Fluorescência por Excitação Multifotônica , Iniciação Traducional da Cadeia Peptídica/genética , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/genética , Retroviridae/genética , Transfecção , Proteínas ViraisRESUMO
UNLABELLED: Wnt/beta-catenin signaling has been proven to play a central role in bone biology. Unexpectedly, the Wnt antagonist Dkk2 is required for terminal osteoblast differentiation and mineralized matrix formation. We show that Dkk1, unlike Dkk2, negatively regulates osteoblast differentiation and bone formation. INTRODUCTION: The Wnt co-receptor LRP5 is a critical regulator of bone mass. Dickkopf (Dkk) proteins act as natural Wnt antagonists by bridging LRP5/6 and Kremen, inducing the internalization of the complex. Wnt antagonists are thus expected to negatively regulation bone formation. However, Dkk2 deficiency results in increased bone, questioning the precise role of Dkks in bone metabolism. MATERIALS AND METHODS: In this study, we investigated specifically the role of Dkk1 in bone in vitro and in vivo. Using rat primary calvaria cells, we studied the effect of retroviral expression of Dkk1 on osteoblast differentiation. In addition, the effect of Dkk1 osteoblast was studied in MC3T3-E1 cells by means of recombinant protein. Finally, to address the role of Dkk1 in vivo, we analyzed the bone phenotype of Dkk1(+/-) animals. RESULTS: Retroviral expression of Dkk1 in rat primary calvaria cells resulted in a complete inhibition of osteoblast differentiation and formation of mineralized nodules, with a marked decrease in the expression of alkaline phosphatase. Dkk1 expression also increased adipocyte differentiation in these cell cultures. Recombinant murine Dkk1 (rmDkk1) inhibited spontaneous and induced osteoblast differentiation of MC3T3-E1 cells. To determine the role of Dkk1 in vivo and overcome the embryonic lethality of homozygous deletion, we studied the bone phenotype in heterozygous Dkk1-deficient mice. Structural, dynamic, and cellular analysis of bone remodeling in Dkk1(+/-) mice showed an increase in all bone formation parameters, with no change in bone resorption, leading to a marked increase in bone mass. Importantly, the number of osteoblasts, mineral apposition, and bone formation rate were all increased several fold. CONCLUSIONS: We conclude that Dkk1 protein is a potent negative regulator of osteoblasts in vitro and in vivo. Given that a heterozygous decrease in Dkk1 expression is sufficient to induce a significant increase in bone mass, antagonizing Dkk1 should result in a potent anabolic effect.
Assuntos
Densidade Óssea/genética , Deleção de Genes , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Osteogênese/genética , Adipogenia/efeitos dos fármacos , Adipogenia/fisiologia , Alelos , Animais , Proteína Morfogenética Óssea 2 , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Diferenciação Celular/efeitos dos fármacos , Regulação para Baixo , Proteínas Hedgehog , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Camundongos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Crânio/citologia , Transativadores/antagonistas & inibidores , Fator de Crescimento Transformador beta/antagonistas & inibidoresRESUMO
BACKGROUND: Restriction enzymes are one of the everyday tools used in molecular biology. The continuously expanding panel of known restriction enzymes (several thousands) renders their optimal use virtually impossible without computerized assistance. Several manufacturers propose on-line sites that assist scientists in their restriction enzyme work, however, none of these sites meet all the actual needs of laboratory workers, and they do not take into account the enzymes actually present in one's own laboratory. RESULTS: Using FileMaker Pro, we developed a stand-alone application which can run on both PCs and Macintoshes. We called it REtools, for Restriction Enzyme tools. This program, which references all currently known enzymes (>3500), permits the creation and update of a personalized list of restriction enzymes actually available in one's own laboratory. Upon opening the program, scientists will be presented with a user friendly interface that will direct them to different menus, each one corresponding to different situations that restriction enzyme users commonly encounter. We particularly emphasized the ease of use to make REtools a solution that laboratory members would actually want to use. CONCLUSION: REtools, a user friendly and easily customized program to organize any laboratory enzyme stock, brings a software solution that will make restriction enzyme use and reaction condition determination straightforward and efficient. The usually unexplored potential of isoschizomers also becomes accessible to all, since REtools proposes all possible enzymes similar to the one(s) chosen by the user. Finally, many of the commonly overlooked subtleties of restriction enzyme work, such as methylation requirement, unusual reaction conditions, or the number of flanking bases required for cleavage, are automatically provided by REtools.
Assuntos
Enzimas de Restrição do DNA/química , Enzimas de Restrição do DNA/classificação , Sistemas de Gerenciamento de Base de Dados , Bases de Dados de Proteínas , Armazenamento e Recuperação da Informação/métodos , Software , Interface Usuário-Computador , Enzimas de Restrição do DNA/análiseRESUMO
BACKGROUND: Internal Ribosome Entry Site (IRES)-based bicistronic vectors are important tools in today's cell biology. Among applications, the expression of two proteins under the control of a unique promoter permits the monitoring of expression of a protein whose biological function is being investigated through the observation of an easily detectable tracer, such as Green Fluorescent Protein (GFP). However, analysis of published results making use of bicistronic vectors indicates that the efficiency of the IRES-controlled expression can vary widely from one vector to another, despite their apparent identical IRES sequences. We investigated the molecular basis for these discrepancies. RESULTS: We observed up to a 10 fold difference in IRES-controlled expression from distinct bicistronic expression vectors harboring the same apparent IRES sequences. We show that the insertion of a HindIII site, in place of the initiating AUG codon of the wild type EMCV IRES, is responsible for the dramatic loss of expression from the second cistron, whereas expression from the first cistron remains unaffected. Thus, while the replacement of the authentic viral initiating AUG by a HindIII site results in the theoretical usage of the initiation codon of the HindIII-subcloned cDNA, the subsequent drop of expression dramatically diminishes the interest of the bicistronic structure. Indeed, insertion of the HindIII site has such a negative effect on IRES function that detection of the IRES-controlled product can be difficult, and sometimes even below the levels of detection. It is striking to observe that this deleterious modification is widely found in available IRES-containing vectors, including commercial ones, despite early reports in the literature stating the importance of the integrity of the initiation codon for optimal IRES function. CONCLUSION: From these observations, we engineered a new vector family, pPRIG, which respects the EMCV IRES structure, and permits easy cloning, tagging, sequencing, and expression of any cDNA in the first cistron, while keeping a high level of expression from its IRES-dependent second cistron (here encoding eGFP).
Assuntos
Engenharia Genética/métodos , Vetores Genéticos/genética , Rim/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Retroviridae/genética , Ribossomos/metabolismo , Transfecção/métodos , Regiões 5' não Traduzidas , Linhagem Celular , Genes/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/genética , Ribossomos/genéticaRESUMO
Expression of the lama3 gene, encoding the laminin alpha3A chain, is restricted to specialized epithelia. We previously showed that lama3 gene expression is controlled by an epithelial enhancer through the cooperative effect of AP-1 binding sites. In fibroblasts, there is no lama3 expression because of the recruitment of a repressor complex absent or inactive in epithelial cells. In this paper, we show evidence that this repression of the lama3 gene is relieved by exogenous and UV-induced USF-1 through its interaction with a non-canonical E-box site. Using a chromatin immunoprecipitation assay, we find that UV stress induces USF to bind to the lama3 promoter in vivo. We further demonstrate that this loss of cell specificity is directly related to the accessibility of the E-box, resulting in a strong induction in fibroblasts, while expression remains constitutively high in keratinocytes. This accessibility appears to be dependent upon the recruitment of a fibroblastic repressor complex. Therefore, we speculate that anchorage of this repressor complex in fibroblasts modifies the enhancer geometry, allowing USF to interact under stress-inducing conditions with its heptameric binding site.
Assuntos
Laminina/genética , Elementos de Resposta , Fatores de Transcrição/metabolismo , Ativação Transcricional , Células 3T3 , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Linhagem Celular Transformada , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/metabolismo , Humanos , Queratinócitos/metabolismo , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Proteínas Repressoras/metabolismo , Raios Ultravioleta , Fatores Estimuladores UpstreamRESUMO
Suicide gene-therapy strategies are promising approaches in treating various diseases such as cancers, atherosclerosis, and graft-versus-host-disease. Here, we describe the development of a new effector gene based on inducing functional caspase 8, the initiator caspase in the death-receptor pathway. We constructed vectors encoding a constitutively active form of human caspase 8 (CC8), and demonstrated the efficient killing of a variety of cell types in transfection and lentivirus-transduction assays. We then analyzed the ability to control the apoptotic activity of a caspase 8-derived construct through the ARIADtrade mark homodimerization system (FKC8), a system shown to be extremely effective in several cellular models upon retroviral and lentiviral gene transfer. Similarly, two transcription-regulation systems, muristerone-regulated and Tet-On, were tested to control the expression of CC8. The homodimerization-regulated system FKC8 was shown to be the most efficient system with low background activity in noninduced conditions. In the presence of a dimerizer, it was as active as the activated Tet-On system. From our data, we conclude that the dimerizer-dependent human caspase 8 represents a highly inducible and very powerful system to eradicate transduced cell populations. In addition to its application in experimental gene therapy, this variant may be highly useful for mechanistic research related to apoptosis.
Assuntos
Apoptose/fisiologia , Caspases/genética , Regulação da Expressão Gênica , Genes Transgênicos Suicidas/genética , Vetores Genéticos , Plasmídeos/genética , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/terapia , Caspase 8 , Caspases/metabolismo , Linhagem Celular , Dimerização , Ecdisterona/análogos & derivados , Ecdisterona/farmacologia , Terapia Genética , Humanos , Lentivirus/genética , Camundongos , Plasmídeos/metabolismo , Retroviridae/genética , Tetraciclina/farmacologia , TransfecçãoRESUMO
In accordance with the two-step hypothesis of T cell activation and the observation that stimulation through the T cell receptor (TCR) alone may lead to anergy, we focused on the introduction of co-stimulatory signaling to this type of receptors to achieve optimal activation. Enhanced mRNA and cell surface receptor expression via the co-stimulatory gene fragment (OX40) was confirmed by RT-PCR and flow cytometry. Inclusion of the OX40 co-stimulatory signaling region in series with the TCR led to enhanced antigen-induced IL-2 production after stimulation by MUC1-expressing cancer cell lines as compared to the chimeric receptor without OX40. Moreover, with the aim of maintaining high efficiency, while providing a means of controlling any possible unwanted proliferation in vivo, a regulation system was used. This controls the dimerization of a membrane-bound caspase 8 protein. Toward that goal, pFKC8 and CAR constructs were co-transfected into Jurkat cells, and the level of apoptosis was measured. 24 h after addition of the dimerizer, a 91% decrease in transfected cells was observed.
Assuntos
Apoptose , Caspase 8/imunologia , Mucina-1/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/citologia , Animais , Caspase 8/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Interleucina-2/imunologia , Ativação Linfocitária , Camundongos , Mucina-1/genética , Células NIH 3T3 , RNA Mensageiro/genética , Receptores de Antígenos de Linfócitos T/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , TransfecçãoRESUMO
Extracellular signal-related kinase (ERK) is a key player in cell signaling. After 25 years of investigation, ERK has been associated with every major aspect of cell physiology. Cell proliferation, cell transformation, protection against apoptosis, among others, are influenced by ERK function. Surprisingly, ERK has also been associated with two apparently opposing processes. The involvement of ERK in cell proliferation has been extensively described, as well as its function in postmitotic cells undergoing differentiation. The analysis of these apparent discrepancies has led to a more precise understanding of the multiple functions and regulations of ERK. More recently, several groups have identified a new and unexpected role for ERK. Although being accepted as an important player in the protection against cell death by apoptosis, it is now clear that ERK can also be directly linked to cell death signaling. Here, we review the role of ERK in cell response to cadmium and its association with cell toxicity. In this system, ERK is subjected to a continuous activation that can last for days, which ultimately results in cell death. Cadmium entry into cells is responsible for this sustained ERK activation, probably via reactive oxygen species production, and protein kinase C has a negative action on this cadmium-dependent ERK activation by modulating cadmium entry into cells.
Assuntos
Cádmio/toxicidade , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Autofagia/efeitos dos fármacos , Autofagia/fisiologia , Cádmio/farmacocinética , Cálcio/metabolismo , Ativação Enzimática/efeitos dos fármacos , Humanos , Transporte de Íons , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Modelos Biológicos , Necrose/induzido quimicamente , Necrose/enzimologia , Proteína Quinase C/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Extracellular signal-related kinase (ERK) is a well-known kinase taking part in a signal transduction cascade in response to extracellular stimuli. ERK is generally viewed as a kinase that is rapidly and transiently phosphorylated following mitogenic stimulation. This activation results in ERK phosphorylating further downstream targets, thus transmitting and amplifying the original stimulus, and ultimately resulting in the onset of cellular proliferation and/or protection against apoptosis. More recently, several groups have identified a strikingly new type of ERK activation that results in cell death. This activation is very different from conventional ERK activation, as it occurs several hours after the original stimulation, and results in the sustained phosphorylation of ERK, which can be observed for up to several days. One way of inducing this delayed ERK activation is by low-dose cadmium treatment. We show here that sustained ERK activation induced by cadmium in human kidney-derived cells is inhibited following protein kinase C (PKC) activation, even when this activation occurs hours before intoxication. Furthermore, PKC inhibition results in an enhanced ERK activation in response to cadmium, even when inhibition is induced hours before intoxication. PKCepsilon appears to be the most implicated isotype in this phenomenon. Finally, we present evidence suggesting that the ZIP8 transporter is involved in this process, as multiple small interfering RNAs against ZIP8 have a protective effect against cadmium treatment. Our results indicate that PKC activation negatively affects ZIP8 transporter activity, thus protecting cells against cadmium poisoning.
Assuntos
Cádmio/toxicidade , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteína Quinase C/metabolismo , Cádmio/farmacocinética , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/fisiologia , Regulação para Baixo , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Humanos , Rim/efeitos dos fármacos , Rim/enzimologia , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Cadmium poisoning has been known to result in a wide variety of cellular responses, including oxidative stress and kinase activation. It has been reported that ERK is activated following acute cadmium exposure, and this response is commonly seen as a classical ERK survival mechanism. Here, we analyzed different cell types for their responses to low concentrations of cadmium poisoning. We found that there is an association between cell susceptibility to cadmium toxicity and ERK activation. This activation is atypical, since it consists of a sustained ERK phosphorylation, that lasts up to 6 days post stimulation. This activation is associated with the appearance of cleaved caspases 8 and 3, processed PARP, and irreversible damage. Pharmacological inhibition of ERK phosphorylation results in the ability of cells to resist cadmium poisoning. Our data indicate that low cadmium concentrations result in an unconventional ERK sustained phosphorylation, which in turn leads to death signaling.
Assuntos
Intoxicação por Cádmio/enzimologia , Intoxicação por Cádmio/patologia , Caspases/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Osteoblastos/enzimologia , Osteoblastos/patologia , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , RatosRESUMO
Cadmium poisoning results in cell death. Although several intracellular pathways have been identified in this response, transport systems responsible for cadmium entry into cells remain poorly understood and controversial. Here, we analyzed the effects of several divalent cations on cadmium toxicity in different cell types. We found that zinc, previously reported as a protective agent against cadmium poisoning, is actually much less efficient than manganese. We show that manganese dramatically reduces cadmium intake, and that this is associated with the inhibition of our recently reported sustained activation of ERK, characteristic of cadmium intoxication. Finally, we show that this inhibition of cadmium entry and ERK-sustained activation perfectly correlates with a high cellular resistance to cadmium exposure. Our results, together with previously published data, support the idea that the yet to be characterized manganese transporter system(s) may be responsible for cadmium entry into cells.
Assuntos
Cádmio/administração & dosagem , Sobrevivência Celular/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/patologia , Manganês/administração & dosagem , Animais , Intoxicação por Cádmio/patologia , Células Cultivadas , Relação Dose-Resposta a Droga , Combinação de Medicamentos , CamundongosRESUMO
In this study, we investigated the effects of Ets2 expression on the proliferation, maturation, and survival of thymocytes by establishing transgenic mice that specifically express Ets2 or a dominant negative form of Ets2, Deltaets2, in the thymus. We show that, in young animals, there are fewer T cells in Deltaets2 transgenic thymi and that the maturation of these T cells is affected at the CD4(-)CD8(-) double-negative to CD4(+)CD8(+) double-positive transition compared with wild-type littermate mice. Partial recovery in the number of thymocytes and full T cell maturation are restored with increasing age of Deltaets2 transgenic animals. However, thymocytes from adult Deltaets2 transgenic mice cultured ex vivo are more sensitive to cell death and to glucocorticoid-induced apoptosis than are T cells from control littermate mice. We also show that T cells from adult ets2 transgenic mice proliferate faster than their wild-type littermates. The proliferation and survival of these T cells are clearly affected upon apoptotic signals: glucocorticoid-induced apoptosis induces T cells from ets2 transgenic mice to continue to proliferate in vivo and to survive better ex vivo than T cells from control littermates. It has been shown that c-Myc expression is required for thymic proliferation and improves thymocyte survival of dexamethasone-treated animals. We show that the expression of c-Myc, an Ets2 target, is elevated in T cells freshly isolated from thymi of ets2 transgenic mice pretreated with dexamethasone. Together, these results show that Ets2 plays a role in the proliferation and survival of thymocytes, implicating a Myc-dependent pathway.
Assuntos
Proteínas de Ligação a DNA , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Repressoras , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Timo/citologia , Timo/imunologia , Transativadores/fisiologia , Fatores de Transcrição , Animais , Animais Recém-Nascidos/genética , Animais Recém-Nascidos/imunologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Apoptose/imunologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Divisão Celular/imunologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Células Cultivadas , Dexametasona/farmacologia , Feminino , Genes myc/efeitos dos fármacos , Genes myc/imunologia , Humanos , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Proto-Oncogênica c-ets-2 , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Timo/efeitos dos fármacos , Transativadores/biossíntese , Transativadores/genética , Transgenes/efeitos dos fármacos , Transgenes/imunologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
Obesity is associated with many serious afflictions such as cardiovascular disease, cancer, and diabetes. One of the main cellular systems used to study the underlying physiological and biological processes is the 3T3-L1 preadipocyte differentiation model. However, studies on 3T3-L1 adipocytes are hampered by the fact that genetic modification of mature adipocytes is notoriously difficult. In this report, we evaluated the use of lentivirus-mediated gene transfer into 3T3-L1 mature adipocytes. We demonstrate that quiescent, fully differentiated 3T3-L1 adipocytes as well as 3T3-L1 preadipocytes can be efficiently transduced with HIV-1-derived lentiviral vectors. Upon transduction using LV-PGK-GFP lentiviral vector at 100 ng p24 per 10(5) cells, more than 95% of the 3T3-L1 adipocytes in the culture expressed the GFP reporter gene. There were no overt signs of toxicity or cytopathogenicity in the cultures. Furthermore, modification of undifferentiated preadipocytes did not affect their capacity to differentiate. In addition, insulin-induced glucose uptake was not affected by the procedure. In contrast, adenoviral-mediated gene transfer into 3T3-L1 adipocytes is associated with marked cytopathogenicity. From these data, we conclude that lentiviral vectors are the gene-transfer system of choice for genetic modification of mature adipocytes. The availability of an efficient vector system may stimulate the use of adipose tissue as a target for gene therapy in obesity and other disorders.