A constitutive cytoprotective pathway protects endothelial cells from lipopolysaccharide-induced apoptosis.

Lipopolysaccharide (LPS) has been implicated as the bacterial component responsible for much of the endothelial cell injury/dysfunction associated with Gram-negative bacterial infections. Protein synthesis inhibition is required to sensitize the endothelium to lipopolysaccharide-induced apoptosis, suggesting that a constitutive or inducible cytoprotective protein(s) is required for endothelial survival. We have identified two known endothelial anti-apoptotic proteins, c-FLIP and Mcl-1, the expression of which is decreased markedly in the presence of cycloheximide. Decreased expression of both proteins preceded apoptosis evoked by lipopolysaccharide + cycloheximide. Caspase inhibition protected against apoptosis, but not the decreased expression of c-FLIP and Mcl-1, suggesting that they exert protection upstream of caspase activation. Inhibition of the degradation of these two proteins with the proteasome inhibitor, lactacystin, prevented lipopolysaccharide + cycloheximide-induced apoptosis. Similarly, lactacystin protected against endothelial apoptosis induced by either tumor necrosis factor-alpha or interleukin-1beta in the presence of cycloheximide. That apoptosis could be blocked in the absence of new protein synthesis by inhibition of the proteasome degradative pathway implicates the requisite involvement of a constitutively expressed protein(s) in the endothelial cytoprotective pathway. Finally, reduction of FLIP expression with antisense oligonucleotides sensitized endothelial cells to LPS killing, demonstrating a definitive role for FLIP in the protection of endothelial cells from LPS-induced apoptosis.

Pertussis toxin-sensitive and -insensitive thrombin stimulation of Shc phosphorylation and mitogenesis are mediated through distinct pathways.

Activation of both receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs) result in phosphorylation of the adaptor protein Shc, providing sites of interaction for proteins in downstream signal transduction cascades. The mechanism of Shc phosphorylation and its function in G protein signaling pathways is still unclear. By examining Shc phosphorylation in response to thrombin in two cell lines, we have defined distinct pertussis toxin (PTX)-sensitive and -insensitive mechanisms by which GPCRs can stimulate tyrosine phosphorylation of Shc. By mutating the tyrosines in Shc, we show that the three sites of tyrosine phosphorylation, Y239, Y240, and Y317, are necessary for thrombin signaling in both systems. The SH2 (src homology 2) domain of Shc is also critical for signaling, but not required for phosphorylation of Shc. In both cell types, inhibition of src family member kinases by chemical inhibitors or microinjection block Shc phosphorylation and bromodeoxyuridine (BrdU) incorporation in response to thrombin. However, in the PTX-sensitive thrombin pathway, both betagamma function and the epidermal growth factor receptor (EGFR) are necessary for Shc phosphorylation and BrdU incorporation. In contrast, signaling in the PTX-insensitive pathway is not mediated through betagamma or the EGFR. Thus, while phosphorylation and function of Shc appear to be the same in both thrombin pathways, the mechanism of tyrosine kinase activation proximal to Shc is different. The differences in signaling between the two thrombin pathways may be representative of mechanisms used by other PTX-sensitive and -insensitive GPCRs to mediate specific responses. In addition, transactivation of RTKs may be a manner by which GPCRs can amplify their signal.

Bifurcation of cell migratory and proliferative signaling by the adaptor protein Shc.

Cytokines and extracellular matrix proteins initiate signaling cascades that regulate cell migration and proliferation. Evidence is provided that the adaptor protein Shc can differentially regulate these processes. Specifically, under growth factor-limiting conditions, Shc stimulates haptotactic cell migration without affecting anchorage-dependent proliferation. However, when growth factors are present, Shc no longer influences cell migration; rather, Shc is crucial for DNA synthesis. Mutational analysis of Shc demonstrates that, while tyrosine phosphorylation is required for both DNA synthesis and cell migration, the switch in Shc signaling is associated with differential use of Shc's phosphotyrosine interacting domains; the PTB domain regulates haptotaxis, while the SH2 domain is selectively required for proliferation.

Evaluating the delivery of nebulized and metered-dose inhalers in an in vitro infant ventilator lung model.

OBJECTIVE: To evaluate drug delivery to the lungs of nebulized and metered-dose inhalers (MDIs) in an in vitro infant lung model. METHODS: An in vitro lung model was modified to study drug delivery. A 1000 mL intravenous bag filled with 500 mL deionized water was attached to a 3.5 mm (12 cm length) endotracheal tube. An inline Marquest Whisper Jet infant circuit nebulizer system delivered 2.5 mg/3 mL albuterol sulfate inhalation solution (Ventolin nebules) at a flow rate of 5 L/min. An Aerochamber (Monaghan) was placed at the endotracheal tube for the delivery of the MDIs. Albuterol MDI (Ventolin) 10 inhalations and beclomethasone MDI (Beclovent) 20 inhalations were delivered. A Servo 900C (Siemens-Elma) was used at the following ventilator settings: positive inspiratory pressure 30 cm H2O), intermittent mandatory ventilation 40 breaths/min, positive end expiratory pressure 4 cm H2O, inspiratory time 0.4 sec. Each formulation was run at least 10 times and assayed in duplicate by HPLC. An unpaired Student's t-test was used to analyze the statistical significance of the data. RESULTS: There was a significantly greater percentage of drug delivery with MDI albuterol (1.96 +/- 0.50) as compared with nebulized albuterol (1.26 +/- 0.37) (p = 0.002) or beclomethasone diproprionate (0.51 +/- 0.24) (p = 0.001). CONCLUSIONS: Albuterol MDI provides a more efficient delivery of drug to the lung as compared with nebulized albuterol and MDI beclomethasone diproprionate.

The G12 coupled thrombin receptor stimulates mitogenesis through the Shc SH2 domain.

Our previous studies in 1321N1 astrocytoma cells demonstrate that thrombin stimulates Ras-dependent mitogenesis through the pertussis toxin insensitive G protein G12. While the direct effectors of G12 are unknown, G12 can transform fibroblasts, utilize Ras and Rac dependent signaling pathways and stimulate GTP loading of Ras. Here we have examined the role of the Shc adaptor protein in mitogenic signaling by the thrombin receptor in 1321N1 cells. As has been reported in other systems, thrombin stimulation results in tyrosine phosphorylation of Shc in 1321N1 cells. We also show that transient expression of G alpha12 results in tyrosine phosphorylation of Shc, thereby identifying Shc as the most proximal G12 effector to date. In addition, we demonstrate by microinjection that thrombin stimulated mitogenesis requires Shc and occurs specifically through the Shc SH2 domain. Expression of the SH2 domain of Shc also inhibits G alpha12 mediated induction of an AP-1 dependent reporter gene demonstrating that G12 utilizes Shc to propagate downstream signals. Our data indicate that Shc is essential for stimulation of Ras dependent mitogenesis and gene expression by the G12 coupled thrombin receptor.

Functional roles of the Shc phosphotyrosine binding and Src homology 2 domains in insulin and epidermal growth factor signaling.

Shc is involved in the activation of Ras in response to many growth factors. Shc contains two phosphotyrosine binding domains, an Src homology 2 (SH2) domain in the carboxyl terminus of the protein and a phosphotyrosine binding (PTB) domain in the amino terminus. Since functional roles for these two domains have not been established, we microinjected glutathione S-transferase fusion proteins of either the Shc PTB or SH2 domains into fibroblasts expressing insulin and epidermal growth factor receptors and measured their effects on DNA synthesis. We found that the Shc PTB was necessary for insulin-induced mitogenic signaling, whereas the SH2 domain was not. In contrast, for epidermal growth factor signaling, the Shc SH2 was functionally more important. These differential modes of signal transduction may be an important factor in determining the specificity of the response of a cell to external stimuli.

Differential expression of a phosphoepitope at the kinetochores of moving chromosomes.

A phosphorylated epitope is differentially expressed at the kinetochores of chromosomes in mitotic cells and may be involved in regulating chromosome movement and cell cycle progression. During prophase and early prometaphase, the phosphoepitope is expressed equally among all the kinetochores. In mid-prometaphase, some chromosomes show strong labeling on both kinetochores; others exhibit weak or no labeling; while in other chromosomes, one kinetochore is intensely labeled while its sister kinetochore is unlabeled. Chromosomes moving toward the metaphase plate express the phosphoepitope strongly on the leading kinetochore but weakly on the trailing kinetochore. This is the first demonstration of a biochemical difference between the two kinetochores of a single chromosome. During metaphase and anaphase, the kinetochores are unlabeled. At metaphase, a single misaligned chromosome can inhibit further progression into anaphase. Misaligned chromosomes express the phosphoepitope strongly on both kinetochores, even when all the other chromosomes of a cell are assembled at the metaphase plate and lack expression. This phosphoepitope may be involved in regulating chromosome movement to the metaphase plate during prometaphase and may be part of a cell cycle checkpoint by which the onset of anaphase is inhibited until complete metaphase alignment is achieved.

Extracellular glutathione is a source of cysteine for cells that express gamma-glutamyl transpeptidase.

We show that gamma-glutamyl transpeptidase (GGT) is a glutathionase that enables cells to use extracellular glutathione as a source of cysteine. We transfected NIH/3T3 mouse fibroblasts with a plasmid containing cDNA for human GGT, and obtained stably transformed cell lines that expressed GGT in its proper orientation on the outer surface of the cell. NIH/3T3 fibroblasts require cysteine for growth and are unable to use extracellular glutathione as a source of cysteine. We demonstrate GGT-positive fibroblasts are able to grow in cysteine-free medium supplemented with glutathione. Cysteine derived from the cleavage of extracellular glutathione can be used to maintain intracellular levels of glutathione. GGT-positive NIH/3T3 cells were able to replenish intracellular glutathione when incubated in cysteine-free medium containing glutathione. GGT-negative cells could not. Therefore, GGT is a glutathionase that provides the cell with access to a secondary source of cysteine.