RESUMO
Metabolic processes result in the release and exchange of H and O atoms from organic material as well as some inorganic salts and gases. These fluxes of H and O atoms into intracellular water result in an isotopic gradient that can be measured experimentally. Using isotope ratio mass spectroscopy, we revealed that slightly over 50% of the H and O atoms in the intracellular water of exponentially-growing cultured Rat-1 fibroblasts were isotopically distinct from growth medium water. We then employed infrared spectromicroscopy to detect in real time the flux of H atoms in these same cells. Importantly, both of these techniques indicate that the H and O fluxes are dependent on metabolic processes; cells that are in lag phase or are quiescent exhibit a much smaller flux. In addition, water extracted from the muscle tissue of rats contained a population of H and O atoms that were isotopically distinct from body water, consistent with the results obtained using the cultured Rat-1 fibroblasts. Together these data demonstrate that metabolic processes produce fluxes of H and O atoms into intracellular water, and that these fluxes can be detected and measured in both cultured mammalian cells and in mammalian tissue.
Assuntos
Fibroblastos/química , Hidrogênio/química , Músculo Esquelético/química , Oxigênio/química , Água/química , Animais , Linhagem Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Hidrogênio/metabolismo , Masculino , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Oxigênio/metabolismo , Ratos , Ratos Sprague-Dawley , Água/metabolismoRESUMO
Cytochrome P450 (P450) 3A4 (CYP3A4) is the most abundant P450 protein in human liver and intestine and is highly inducible by a variety of drugs and other compounds. The P450 catalytic cycle is known to uncouple and release reactive oxygen species (ROS), but the effects of ROS from P450 and other enzymes in the endoplasmic reticulum have been poorly studied from the perspective of effects on cell biology. In this study, we expressed low levels of CYP3A4 in HepG2 cells, a human hepatocarcinoma cell line, and examined effects on intracellular levels of ROS and on the secretion of a variety of growth factors that are important in extracellular communication. Using the redox-sensitive dye RedoxSensor red, we demonstrate that CYP3A4 expression increases levels of ROS in viable cells. A custom ELISA microarray platform was employed to demonstrate that expression of CYP3A4 increased secretion of amphiregulin, intracellular adhesion molecule 1, matrix metalloprotease 2, platelet-derived growth factor (PDGF), and vascular endothelial growth factor, but suppressed secretion of CD14. The antioxidant N-acetylcysteine suppressed all P450-dependent changes in protein secretion except for CD14. Quantitative RT-PCR demonstrated that changes in protein secretion were consistently associated with corresponding changes in gene expression. Inhibition of the NF-κB pathway blocked P450 effects on PDGF secretion. CYP3A4 expression also altered protein secretion in human mammary epithelial cells and C10 mouse lung cells. Overall, these results suggest that increased ROS production in the endoplasmic reticulum alters the secretion of proteins that have key roles in paracrine and autocrine signaling.
Assuntos
Comunicação Autócrina , Comunicação Parácrina , Espécies Reativas de Oxigênio/metabolismo , Animais , Antioxidantes/metabolismo , Células Cultivadas , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Células Epiteliais/metabolismo , Células Hep G2 , Humanos , Pulmão/citologia , Pulmão/metabolismo , Camundongos , NF-kappa B/metabolismo , OxirreduçãoRESUMO
Transactivation of the epidermal growth factor receptor (EGFR) is thought to be a process by which a variety of cellular inputs can be integrated into a single signaling pathway through either stimulated proteolysis (shedding) of membrane-anchored EGFR ligands or by modification of the activity of the EGFR. As a first step towards building a predictive model of the EGFR transactivation circuit, we quantitatively defined how signals from multiple agonists were integrated both upstream and downstream of the EGFR to regulate extracellular signal regulated kinase (ERK) activity in human mammary epithelial cells. By using a "non-binding" reporter of ligand shedding, we found that transactivation triggers a positive feedback loop from ERK back to the EGFR such that ligand shedding drives EGFR-stimulated ERK that in turn drives further ligand shedding. Importantly, activated Ras and ERK levels were nearly linear functions of ligand shedding and the effect of multiple, sub-saturating inputs was additive. Simulations showed that ERK-mediated feedback through ligand shedding resulted in a stable steady-state level of activated ERK, but also showed that the extracellular environment can modulate the level of feedback. Our results suggest that the transactivation circuit acts as a context-dependent integrator and amplifier of multiple extracellular signals and that signal integration can effectively occur at multiple points in the EGFR pathway.
Assuntos
Células Epiteliais/metabolismo , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Transdução de Sinais/fisiologia , Linhagem Celular , Relação Dose-Resposta a Droga , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Flavonoides/farmacologia , Gefitinibe , Redes Reguladoras de Genes/efeitos dos fármacos , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Imidazóis/farmacologia , Ligantes , Lisofosfolipídeos/farmacologia , Glândulas Mamárias Humanas/citologia , Fosforilação/efeitos dos fármacos , Piridinas/farmacologia , Quinazolinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Ativação Transcricional/efeitos dos fármacos , Fator de Crescimento Transformador alfa/farmacologiaRESUMO
Although the ERK pathway has a central role in the response of cells to growth factors, its regulatory structure and dynamics are incompletely understood. To investigate ERK activation in real time, we expressed an ERK-GFP fusion protein in human mammary epithelial cells. On EGF stimulation, we observed sustained oscillations of the ERK-GFP fusion protein between the nucleus and cytoplasm with a periodicity of approximately 15 min. The oscillations were persistent (>45 cycles), independent of cell cycle phase, and were highly dependent on cell density, essentially disappearing at confluency. Oscillations occurred even at ligand doses that elicited very low levels of ERK phosphorylation, and could be detected biochemically in both transfected and nontransfected cells. Mathematical modeling revealed that negative feedback from phosphorylated ERK to the cascade input was necessary to match the robustness of the oscillation characteristics observed over a broad range of ligand concentrations. Our characterization of single-cell ERK dynamics provides a quantitative foundation for understanding the regulatory structure of this signaling cascade.
Assuntos
Relógios Biológicos , Fator de Crescimento Epidérmico/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Células Epiteliais , Humanos , Fosforilação , Transdução de SinaisRESUMO
Here we identify the release of annexin A2 into the culture medium in response to low-dose X-radiation exposure and establish functional linkages to an established paracrine factor-mediated anchorage-independent growth response. Using a standard bicameral coculture model, we demonstrate that annexin A2 is secreted into the medium by irradiated cells (seeded in upper chamber) and is capable of binding to nonirradiated neighboring cells (seeded in lower chamber). The paracrine factor-mediated anchorage-independent growth response to low-dose X irradiation is reduced when irradiated annexin A2-silenced (shRNA) JB6 cells are co-cultured with nonirradiated cells relative to co-culture with irradiated annexin A2-competent vector control cells. Consistent with this observation, purified bovine annexin A2 tetramer induces anchorage-independent growth. These observations suggest that annexin A2 regulates, in part, the radiation paracrine factor-specific anchorage-independent growth response in JB6 cells.
Assuntos
Anexina A2/metabolismo , Proliferação de Células/efeitos da radiação , Comunicação Parácrina/efeitos da radiação , Sequência de Aminoácidos , Análise de Variância , Animais , Anexina A2/química , Anexina A2/genética , Western Blotting , Bovinos , Linhagem Celular , Técnicas de Cocultura , Fibrinolisina/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Camundongos , Dados de Sequência Molecular , Plasminogênio/metabolismo , Interferência de RNA , Doses de RadiaçãoRESUMO
Bone-forming osteoblasts and their progenitors are target cells for the lipid growth factor lysophosphatidic acid (LPA) which is produced by degranulating platelets at sites of tissue injury. LPA is a potent inducer of bone cell chemotaxis, proliferation and survival in vitro, and this lipid factor is an attractive candidate to facilitate preosteoblast migration during skeletal regeneration in vivo. In this study we sought to more clearly define the intracellular signaling pathways mediating the effects of LPA on bone cells. LPA-treated MC3T3-E1 preosteoblastic cells exhibited a bimodal activation of extracellular signal-related kinase (ERK1/2) with maximal phosphorylation at 5 and 60 min. MEK1/2 activation was detected within 2.5 min of LPA exposure and remained elevated for at least an hour. ERK1/2 phosphorylation was not coupled to Ras activation or to LPA-induced elevations in cytosolic Ca(2+). While LPA exposure transactivates the EGF receptor in many cell types, LPA-stimulated ERK1/2 activation in MC3T3-E1 cells was unaffected by the inhibition of EGF receptor function. ERK isoforms can function as transcription factors and ERK1/2 rapidly accumulated in the nuclei of LPA-treated cells, a process that was blocked if ERK1/2 phosphorylation was prevented. Blocking ERK1/2 phosphorylation also led to significant decreases in LPA-induced MC3T3-E1 cell chemotaxis, while the inhibition of EGF receptor function had no effect on the stimulation of preosteoblast motility by LPA. Our results identify ERK1/2 activation as a mediator of LPA-stimulated MC3T3-E1 cell migration that may be relevant to preosteoblast motility and gene expression during bone repair in vivo. J. Cell. Physiol. 219: 716-723, 2009. (c) 2009 Wiley-Liss, Inc.
Assuntos
Quimiotaxia/efeitos dos fármacos , Receptores ErbB/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Lisofosfolipídeos/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Células 3T3 , Animais , Sequência de Bases , Movimento Celular/efeitos dos fármacos , Quimiotaxia/fisiologia , Primers do DNA/genética , Ativação Enzimática/efeitos dos fármacos , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Ativação Transcricional/efeitos dos fármacos , TransfecçãoRESUMO
The number of distinct signaling pathways that can transactivate the epidermal growth factor receptor (EGFR) in a single cell type is unclear. Using a single strain of human mammary epithelial cells, we found that a wide variety of agonists, such as lysophosphatidic acid (LPA), uridine triphosphate, growth hormone, vascular endothelial growth factor, insulin-like growth factor-1 (IGF-1), and tumor necrosis factor-alpha, require EGFR activity to induce ERK phosphorylation. In contrast, hepatocyte growth factor can stimulate ERK phosphorylation independent of the EGFR. EGFR transactivation also correlated with an increase in cell proliferation and could be inhibited with metalloprotease inhibitors. However, there were significant differences with respect to transactivation kinetics and sensitivity to different inhibitors. In particular, IGF-1 displayed relatively slow transactivation kinetics and was resistant to inhibition by the selective ADAM-17 inhibitor WAY-022 compared with LPA-induced transactivation. Studies using anti-ligand antibodies showed that IGF-1 transactivation required amphiregulin production, whereas LPA was dependent on multiple ligands. Direct measurement of ligand shedding confirmed that LPA treatment stimulated shedding of multiple EGFR ligands, but paradoxically, IGF-1 had little effect on the shedding rate of any ligand, including amphiregulin. Instead, IGF-1 appeared to work by enhancing EGFR activation of Ras in response to constitutively produced amphiregulin. This enhancement of EGFR signaling was independent of both receptor phosphorylation and PI-3-kinase activity, suggestive of a novel mechanism. Our studies demonstrate that within a single cell type, the EGFR autocrine system can couple multiple signaling pathways to ERK activation and that this modulation of EGFR autocrine signaling can be accomplished at multiple regulatory steps.
Assuntos
Células Epiteliais/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Glândulas Mamárias Humanas/metabolismo , Ativação Transcricional/genética , Linhagem Celular , Ativação Enzimática , Receptores ErbB/agonistas , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Cinética , Ligantes , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Peptídeo Hidrolases/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Especificidade por SubstratoRESUMO
SUMMARY: ProMAT is a software tool for statistically analyzing data from enzyme-linked immunosorbent assay microarray experiments. The software estimates standard curves, sample protein concentrations and their uncertainties for multiple assays. ProMAT generates a set of comprehensive figures for assessing results and diagnosing process quality. The tool is available for Windows or Mac, and is distributed as open-source Java and R code. AVAILABILITY: ProMAT is available at http://www.pnl.gov/statistics/ProMAT. ProMAT requires Java version 1.5.0 and R version 1.9.1 (or more recent versions). ProMAT requires either Windows XP or Mac OS 10.4 or newer versions.
Assuntos
Algoritmos , Ensaio de Imunoadsorção Enzimática/métodos , Análise Serial de Proteínas/métodos , Software , Interface Usuário-Computador , Interpretação Estatística de DadosRESUMO
Protein microarrays are a rapidly developing analytic tool with diverse applications in biomedical research. These applications include profiling of disease markers or autoimmune responses, understanding molecular pathways, protein modifications, and protein activities. One factor that is driving this expanding usage is the wide variety of experimental formats that protein microarrays can take. In this review, we provide a short, conceptual overview of the different approaches for protein microarray. We then examine some of the most significant applications of these microarrays to date, with an emphasis on how global protein analyses can be used to facilitate biomedical research.
Assuntos
Fenômenos Fisiológicos Celulares , Técnicas e Procedimentos Diagnósticos/tendências , Análise Serial de Proteínas/tendências , Humanos , Neoplasias/diagnóstico , Análise Serial de Proteínas/métodosRESUMO
We have previously reported that CYP3A cross-links with polyubiquitinated proteins in microsomes from nicardipine-treated rats in a process that is distinct from classical polyubiquitination. To further examine the role of the proteasome in CYP3A degradation, we investigated the effects of proteasome inhibitors lactacystin, MG132, proteasome inhibitor 1, and hemin in primary cultures of rat and human hepatocytes. With the exception of hemin, these agents increased the total pool of ubiquitinated proteins in microsomes isolated from rat hepatocytes, indicating that lactacystin, MG132, and proteasome inhibitor 1 effectively inhibited the proteasome in these cells. All four agents caused a reduction in the amount of the major approximately 55-kDa CYP3A band, opposite to what would be expected if the ubiquitin-proteasome pathway degraded CYP3A. Only hemin treatment caused an increase in high molecular mass (HMM) CYP3A bands. Because hemin treatment did not alter levels of ubiquitin in CYP3A immunoprecipitates, the HMM CYP3A bands formed in response to hemin treatment clearly were not due to proteasome inhibition. Rather, because hemin treatment also caused an increase in HMM CYP3A in the detergent-insoluble fraction of the 10,000g pellet, the HMM CYP3A seems to represent a large protein complex that is unlikely to primarily represent ubiquitination.