Mechanisms of hypoxia-induced endothelial cell death. Role of p53 in apoptosis.
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Endothelial cell death may contribute to tissue injury from ischemia. Little is known, however, about the characteristics of endothelial cell death in response to hypoxia. Using an in vitro model, we found that human umbilical vein endothelial cells were resistant to hypoxia-induced cell death with only a 2% reduction in viability at 24 h and 45% reduction in viability at 48 h. Overexpression of a mutant, IkappaBalpha, via adenoviral vector did not potentiate cell death in hypoxia, indicating that nuclear factor-kappaB activation was not involved in cytoprotection. Cell death in hypoxia was determined to be apoptotic by 3' labeling of DNA using terminal deoxynucleotidyl transferase staining and reversibility of cell death with a caspase inhibitor. Exposure of endothelial cells to hypoxia did not alter levels of proapoptotic and antiapoptotic Bcl-2 family members Bax and Bcl-XL by immunoblot analysis. In contrast, changes in p53 protein levels correlated with the induction of apoptosis in hypoxic endothelial cells. Inhibition of the proteasome increased p53 protein levels and accelerated cell death in hypoxia. Overexpression of p53 by adenoviral transduction was sufficient to initiate apoptosis of normoxic endothelial cells. These data provide a framework for the study of factors regulating endothelial cell survival and death in hypoxia. (+info)
A leucine-rich nuclear export signal in the p53 tetramerization domain: regulation of subcellular localization and p53 activity by NES masking.
(10/5969)
Appropriate subcellular localization is crucial for regulating p53 function. We show that p53 export is mediated by a highly conserved leucine-rich nuclear export signal (NES) located in its tetramerization domain. Mutation of NES residues prevented p53 export and hampered tetramer formation. Although the p53-binding protein MDM2 has an NES and has been proposed to mediate p53 export, we show that the intrinsic p53 NES is both necessary and sufficient for export. This report also demonstrates that the cytoplasmic localization of p53 in neuroblastoma cells is due to its hyperactive nuclear export: p53 in these cells can be trapped in the nucleus by the export-inhibiting drug leptomycin B or by binding a p53-tetramerization domain peptide that masks the NES. We propose a model in which regulated p53 tetramerization occludes its NES, thereby ensuring nuclear retention of the DNA-binding form. We suggest that attenuation of p53 function involves the conversion of tetramers into monomers or dimers, in which the NES is exposed to the proteins which mediate their export to the cytoplasm. (+info)
High concentration of glucose decreases glucose transporter-1 expression in mouse placenta in vitro and in vivo.
(11/5969)
Facilitative glucose transporter-1 (GLUT1) is expressed abundantly and has an important role in glucose transfer in placentas. However, little is known about the regulation of GLUT1 expression in placental cells. We studied the changes in placental GLUT1 levels in relation to changes in glucose concentration in vitro and in vivo. In in vitro experiments, dispersed mouse placental cells were incubated under control (5.5 mM) and moderately high (22 mM) glucose concentrations, and 2-deoxyglucose uptake into cells was studied on days 1-5 of culture. After 4 days of incubation under both conditions, GLUT1 mRNA and proten levels were examined by Northern and immunoblot analyses. Treatment of cells with 22 mM glucose resulted in a significant decrease in 2-deoxyglucose uptake compared with control, from day 2 to day 5 of culture. Moreover, GLUT1 mRNA and protein levels on day 4 of culture were significantly reduced in cells incubated with 22 mM glucose compared with control. Next, we rendered mice diabetic by administering 200 micrograms/g body weight streptozotocin (STZ) on day 8 of pregnancy. Animals were killed on day 12 of pregnancy and placental tissues were obtained. [3H]Cytochalasin B binding study was carried out to assess total GLUTs, and GLUT1 mRNA and protein were measured as above. [3H]Cytochalasin B binding sites in placentas from STZ-treated mice were significantly less than those in control mice. Northern and immunoblot analyses revealed a significant decrease in GLUT1 mRNA and protein levels in diabetic mice compared with the controls. These findings suggest that the glucose concentration may regulate the expression of placental GLUT1. (+info)
A model of whole-body protein turnover based on leucine kinetics in rodents.
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The measurement of fractional synthesis rate is based on the following assumptions: amino acids for protein synthesis are supplied by an intracellular pool; amino acids from protein degradation are not recycled preferentially to protein synthesis; and proteins turn over at a homogeneous rate. To test these assumptions, a mechanistic, theoretical model of protein turnover for a nongrowing 26-g mouse was developed on the basis of data from the literature. The model consisted of three protein pools turning over at fast (102 micromol Leu, t1/2= 11.5 h), medium (212 micromol Leu, t1/2 = 16.6 h) or slow (536 micromol Leu, t1/2 = 71.5 h) rates and extracellular (1.69 micromol Leu), leucyl-tRNA (0.0226 micromol Leu) and intracellular (5.72 micromol Leu) amino acid pools that exchanged amino acids. The flow of amino acids from the protein pools to the leucyl-tRNA pool determined the amount of recycling. The flow of amino acids from the extracellular pool to aminoacyl tRNA determined the amount of channeling. Two flooding dose data sets were used to evaluate specific radioactivity changes predicted by the model. Predictions of specific radioactivities using flooding dose, pulse dose or continuous infusion methods indicated that the model can be a useful tool in estimating the rates of channeling and recycling. However, it was found that use of data from flooding dose experiments might cause inaccurate predictions of certain fluxes. (+info)
Recycling, channeling and heterogeneous protein turnover estimation using a model of whole-body protein turnover based on leucine kinetics in rodents.
(13/5969)
In the companion paper, a whole-body, mechanistic model of protein turnover in a rodent was described and evaluated with independent data sets that used the flooding dose method. On the basis of fitted fluxes, the model was able to predict specific radioactivity changes in the protein and free leucine pools and whole-body protein fractional synthesis rate (FSR). In this paper, results of model simulations of specific radioactivity changes in the flooding dose, pulse dose and continuous infusion methods were compared and the influence of recycling, channeling and multiple protein pools on model behavior were analyzed. For all methods, the percentage of channeling must be estimated to determine whether the extracellular or intracellular pool specific radioactivities better approximate the aminoacyl tRNA pool specific radioactivity. Recycling also affects the specific radioactivity of the aminoacyl-tRNA pool and therefore must be estimated. An analysis of fits of the flooding dose data indicated that 100% channeling was occurring, but the percentage of recycling could not be determined. Multiple protein pools turning over at different rates overestimated FSR by 2-3% at early time points (5 min) and underestimated FSR by 3-6% at 60 min in the flooding dose method. For the pulse dose method, FSR was underestimated by 40-50% at 5 min and underestimated by 9-10% at 60 min. An increase in time to measure FSR caused a decrease in the estimate of FSR (18% over 3 h) for the flooding dose method and an increase in the estimate of FSR (144% over 3 h) for the pulse dose method. (+info)
Identification of a Leu-lle internalization motif within the cytoplasmic domain of the leukaemia inhibitory factor receptor.
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Leukaemia inhibitory factor (LIF) signals via a heterodimeric receptor complex comprised of the LIF receptor (LIFR) and the interleukin (IL)-6 signal transducer gp130. Upon binding to its cognate receptor LIF is internalized. In this study, we show that the LIFR is endocytosed independently of gp130. By using a heterochimaeric receptor system we identified a dileucine-based internalization motif within the cytoplasmic domain of the LIFR. Our findings suggest that a heterodimeric LIFR/gp130 complex and homodimeric gp130/gp130 complex are endocytosed via distinct internalization signals. (+info)
Metalloproteinases are involved in lipopolysaccharide- and tumor necrosis factor-alpha-mediated regulation of CXCR1 and CXCR2 chemokine receptor expression.
(15/5969)
The neutrophil-specific G-protein-coupled chemokine receptors, CXCR1 and CXCR2, bind with high affinity to the potent chemoattractant interleukin-8 (IL-8). The mechanisms of IL-8 receptor regulation are not well defined, although previous studies have suggested a process of ligand-promoted internalization as a putative regulatory pathway. Herein, we provide evidence for two distinct processes of CXCR1 and CXCR2 regulation. Confocal microscopy data showed a redistribution of CXCR1 expression from the cell surface of neutrophils to internal compartments after stimulation with IL-8, whereas stimulation with bacterial lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-alpha) did not induce CXCR1 internalization but instead mediated a significant loss of membrane-proximal CXCR1 staining intensity. To investigate whether proteolytic cleavage was the mechanism responsible for LPS- and TNF-alpha-induced downmodulation of IL-8 receptors, we tested a panel of proteinase inhibitors. The downmodulation of CXCR1 and CXCR2 by LPS and TNF-alpha was most dramatically inhibited by metalloproteinase inhibitors; 1, 10-phenanthroline and EDTA significantly attenuated LPS- and TNF-alpha-induced loss of CXCR1 and CXCR2 cell surface expression. Metalloproteinase inhibitors also blocked the release of CXCR1 cleavage fragments into the cell supernatants of LPS- and TNF-alpha-stimulated neutrophils. In addition, while treatment of neutrophils with LPS and TNF-alpha inhibited IL-8 receptor-mediated calcium mobilization and IL-8-directed neutrophil chemotaxis, both 1, 10-phenanthroline and EDTA blocked these inhibitory processes. In contrast, metalloproteinase inhibitors did not affect IL-8-mediated downmodulation of CXCR1 and CXCR2 cell surface expression or receptor signaling. Thus, these findings may provide further insight into the mechanisms of leukocyte regulation during immunologic and inflammatory responses. (+info)
A heptad motif of leucine residues found in membrane proteins can drive self-assembly of artificial transmembrane segments.
(16/5969)
Specific interactions between alpha-helical transmembrane segments are important for folding and/or oligomerization of membrane proteins. Previously, we have shown that most transmembrane helix-helix interfaces of a set of crystallized membrane proteins are structurally equivalent to soluble leucine zipper interaction domains. To establish a simplified model of these membrane-spanning leucine zippers, we studied the homophilic interactions of artificial transmembrane segments using different experimental approaches. Importantly, an oligoleucine, but not an oligoalanine, se- quence efficiently self-assembled in membranes as well as in detergent solution. Self-assembly was maintained when a leucine zipper type of heptad motif consisting of leucine residues was grafted onto an alanine host sequence. Analysis of point mutants or of a random sequence confirmed that the heptad motif of leucines mediates self-recognition of our artificial transmembrane segments. Further, a data base search identified degenerate versions of this leucine motif within transmembrane segments of a variety of functionally different proteins. For several of these natural transmembrane segments, self-interaction was experimentally verified. These results support various lines of previously reported evidence where these transmembrane segments were implicated in the oligomeric assembly of the corresponding proteins. (+info)