Receptor-mediated internalization of Pseudomonas toxin by mouse fibroblasts

Pseudomonas exotoxin (PE) was used as a probe to study the mechanism by which protein ligands are internalized by mammalian cells. Both biochemical and electron microscopic methods were used to look at the internalization of PE by mouse LM cell fibroblasts. Our data suggest that PE enters cells by receptor-mediated endocytosis, a process previously thought to be restricted to the entry of biologically significant molecules such as lysosomal enzymes and peptide hormones. Biochemical studies showed that methylamine (20 mM) and chloroquine (10 microM) protected LM cells from the action of PE. Full protection was observed if methylamine or chloroquine was added to the monolayers simultaneously with toxin or if they were added up to 10 min after toxin binding. Later addition of amine or chloroquine afforded partial protection to the monolayers. With immunoelectron microscopy we observed that in the cold toxin bound diffusely to the cell surface but was rapidly internalized when cells were warmed to 37 degrees C. In the presence of methylamine, chloroquine or ammonium chloride, internalization did not occur. We propose that PE enters mouse fibroblasts by receptor-mediated endocytosis and that chloroquine and methylamine, agents which are known to block this process, prevent expression of toxicity.     

Phagosomal membrane lipids of LM fibroblasts

Summary Murine fibroblasts, LM cells, were cultured in suspension or monolayer in a chemically defined medium without serum and exposed to polystyrene beads. The LM cells endocytized the beads in direct proportion to the bead/cell ratio and the bead surface area. However, equal volumes of beads irrespective of size or surface area were internalized. The lipid composition of the phagosome membrane differed significantly from the parent primary membrane in having higher contents of phosphatidylcholine, phosphatidylserine, and sterol but lower contents of sphingomyelin and lysophosphatidylcholine. When phagosomes isolated from suspension-cultured LM fibroblasts were exposed to trinitrobenzenesulfonic acid at 4°C, 55±1.6% of the phagosomal membrane phosphatidylethanolamine was trinitrophenylated. The asymmetric distribution of phosphatidylethanolamine across the phagosomal membrane was not affected by the bead/cell ratio, bead diameter, or exposure time of LM fibroblasts to the beads. When cells were reacted with trinitrobenzenesulfonic acid at 4°C prior to phagocytosis, the amount of trinitrophenylphosphatidylethanolamine was greater in the isolated phagosomes than in the parent primary plasma membrane. Culturing LM fibroblasts in suspension or monolayer had no effect on the asymmetric distribution of phosphatidylethanolamine across primary plasma membrane bilayers. The data are consistent with the observation that LM fibroblasts grown either in suspension or monolayer internalize polystyrene beads at selective sites in the surface membrane.     

Calmodulin antagonists sensitize cells to pseudomonas toxin

L cells and mouse 3T3 cells, which are very sensitive to Pseudomonas aeruginosa exotoxin A (PEA), were protected with weak bases and low concentrations of monensin. BHK cells and a number of other cell lines which are much less sensitive to PEA were much less protected under these conditions. Trifluoperazine, dansylcadaverine, and several other calmodulin antagonists strongly sensitized BHK cells to the toxin whereas they did not affect the sensitivity of the mouse 3T3 and L cells. The sensitization of the BHK cells was counteracted by treatment with weak bases or low concentrations of monensin. Calmodulin antagonists also sensitized cells to toxin which had become inaccessible to antitoxin, indicating that the effect of the calmodulin antagonists is exerted on a process taking place after the toxin is endocytosed.     

Altered endocytosis in a mutant of LM fibroblasts defective in cell–cell fusion

Mutants of LM fibroblasts selected for their decreased ability to undergo polyethylene glycol-induced cell-to-cell fusion (F40 subline) were examined for possible alterations of their ability to carry out endocytosis. Both fluidphase endocytosis of inulin and horseradish peroxidase and nonreceptor mediated adsorptive endocytosis of poly(L-lysine) were reduced to 60% of control values. Comparable results were obtained when the uptake of poly(L-lysine) was measured as internalization of surface-bound label in label-free medium or following continuous exposure. Accelerated breakdown of internalized label was ruled out as a cause for decreased label accumulation. Accelerated exocytosis is an unlikely cause, and it is suggested that the decreased uptake is due to a decrease in the constitutive membrane vesiculation process that leads to the formation of endocytotic vesicles. The capacity of F40 cells to degrade internalized horseradish peroxidase and poly(L-lysine) was not impaired, nor was their susceptibility to the cytotoxic action of methotrexate-poly(L-lysine). This drug conjugate must be degraded inside cells and release small molecular methotrexate in order to be cytocidal. These data suggest that only the first step of nonspecific endocytosis is impaired, while the subsequent steps that require fusion of endosomes to lysosomes proceed normally. Since the formation of primary endosomes requires membrane fusion through the external aspect of the plasma membrane and in that respect resembles cell-cell fusion, we propose the hypothesis that the observed decrease in endocytosis is related to the decreased ability of F40 cells to fuse with each other, and reflects a decreased efficiency of fusion processes at the external face of the plasma membrane.     

Barnase toxin: a new chimeric toxin composed of pseudomonas exotoxin A and barnase

We have constructed a chimeric toxin composed of Pseudomonas exotoxin A (PE) and the extracellular ribonuclease of Bacillus amyloliquefaciens, barnase. The chimeric protein, termed PE-Bar, reacted with both anti-PE and anti-barnase antisera and had both ADP ribosylation and ribonuclease activities. The chimeric toxin was cytotoxic to the murine fibroblast cell line L929 and to a murine hybridoma resistant to PE. A mutant form of PE-Bar lacking ADP-ribosylating activity was still cytotoxic to L929 cells. Because treatment of cells prelabeld with [3H]uridine resulted in a decrease in their RNA content, we conclude that this cytotoxic effect was due to the ribonuclease activity of barnase molecules that had been translocated to the cytosol. It is now possible to construct chimeric toxins with two or more enzymatic activities that can be delivered to the cytosol of the target cells.     

Effect of potassium depletion of cells on their sensitivity to diphtheria toxin and pseudomonas toxin

When Vero cells were depleted of potassium, the cells were protected against diphtheria toxin. Potassium depletion of Vero cells strongly reduced the binding of the toxin to cell surface receptors. Likewise, potassium depleted L-cells were protected against pseudomonas toxin. Diphtheria toxin binding was completely restored upon addition of potassium to the cells. This restoration was not prevented by inhibition of protein synthesis by cycloheximide. When cells were depleted of potassium in the presence of metabolic inhibitors, and then treated with diphtheria toxin, protein synthesis was reduced to the same extent as in cells with normal intracellular level of potassium. The results indicate that potassium depletion of Vero cells reduces the ability of the cells to bind diphtheria toxin by an ATP requiring process, and that binding, endocytosis and transfer of diphtheria fragment A across the membrane may occur at low intracellular levels of potassium.     

PEDF from mouse mesenchymal stem cell secretome attracts fibroblasts

Conditioned medium (secretome) derived from an enriched stem cell culture stimulates chemotaxis of human fibroblasts. These cells are classified as multipotent murine mesenchymal stromal cells (mMSC) by immunochemical analysis of marker proteins. Proteomic analysis of mMSC secretome identifies nineteen secreted proteins, including extracellular matrix structural proteins, collagen processing enzymes, pigment epithelium-derived factor (PEDF) and cystatin C. Immunodepletion and reconstitution experiments show that PEDF is the predominant fibroblast chemoattractant in the conditioned medium, and immunofluorescence microscopy shows strong staining for PEDF in the cytoplasm, at the cell surface, and in intercellular space between mMSCs. This stimulatory effect of PEDF on fibroblast chemotaxis is in contrast to the PEDF-mediated inhibition of endothelial cell migration, reported previously. These differential functional effects of PEDF toward fibroblasts and endothelial cells may serve to program an ordered temporal sequence of scaffold building followed by angiogenesis during wound healing.     

Effects of amiloride on hyperthermic cell killing of normal and thermotolerant mouse fibroblast LM cells

The effect of amiloride on hyperthermic cell killing of normal and thermotolerant mouse fibroblast LM cells was investigated under normal (pH 7.4) and acidic (pH 6.8) conditions. Amiloride is known to inhibit the Na+/H+ exchanger in the plasma membrane, the main pH regulating mechanism in mammalian cells. The effects of low pH and amiloride on the mouse fibroblasts were qualitatively similar. For normal cells, mainly a reduction of the shoulder of the survival curve was observed, while an increase of the slope of the exponential part of the survival curve was found in thermotolerant cells. When a combination of 3 mmol dm-3 amiloride and low pH was used the effect on the hyperthermic sensitivity of normal and thermotolerant cells was not additive. This may be explained by a similarity in the mechanism of action of the two treatments, viz. inhibition of the Na+/H+ exchange, which is probably complete when 3 mmol dm-3 amiloride is used. The amiloride sensitivity of normal and thermotolerant fibroblasts is dose dependent in the range of 0.1 to 3 mmol dm-3. Because the D0 of control cells is almost independent of the amount of amiloride, a concentration-dependent reduction of the thermotolerance ratio is found, especially at higher concentrations of amiloride.     

JSAP1 is required for the cell adhesion and spreading of mouse embryonic fibroblasts

The roles of JSAP1 and JIP1 in cell adhesion and spreading were examined using mouse embryonic fibroblasts (MEFs) deficient in JIP1 (JIP1-KO), JSAP1 (JSAP1-KO), and in both JIP1 and JSAP1 (double-KO), and by using their wild type. After being plated on fibronectin-coated culture plates, wild type MEFs rapidly adhered and differentiated to typical longitudinal fibroblasts in 4 h. JSAP1-KO MEFs showed a similar sequence of adhesion and cell spreading, but their adhesion was weak, and cell spreading sequence proceeded in a delayed manner compared with the wild type. In spreading JSAP1-KO MEFs, adhesion-triggered actin cytoskeleton reorganization and FAK activation proceeded at a slower pace than in wild type MEFs. The cellular properties of double-KO MEFs and JIP1-KO MEFs were similar to those of JSAP1-KO MEFs and wild type MEFs, respectively. These results suggest that JSAP1 plays a role in adhesion and cell spreading by regulating the rapid reorganization of the actin cytoskeleton.     

Toxicity of oxidized low-density lipoprotein to cultured fibroblasts is selective for S phase of the cell cycle

Oxidized LDL (o-LDL) is toxic to a variety of cultured cells. Preliminary results suggested that susceptibility is enhanced by cell proliferation. As a step toward determining the mechanism of cytotoxicity, we chose to identify the cell cycle phase(s) during which exposure of cultured human fibroblasts to o-LDL leads to death. Cytochalasin B, which blocks cell migration and proliferation, and irradiation, which prevents mitosis but not migration, both blocked cytotoxicity. Colchicine, which arrests cells in mitosis but does not inhibit DNA synthesis, did not block cytotoxicity. Treatment of cells with hydroxyurea, which blocks cells prior to S phase, prevented cell death. Addition of o-LDL to cells immediately after S phase allowed mitosis without death. The above results coupled with results using cells synchronized by three different means indicate that cell death is selective for proliferating cells and occurs after exposure to o-LDL during S phase. Understanding the mechanism of o-LDL-induced death may have implications for tissue damage in vivo in the numerous instances of pathology in which oxidized lipoproteins or lipids are present.     

Identification of superoxide dismutase as a cofactor for the pseudomonas type III toxin, ExoU

Pseudomonas aeruginosa is an opportunistic pathogen that uses a type III secretion system and four effector proteins to avoid innate immune responses. ExoS, ExoT, ExoY, and ExoU all possess enzymatic activities that disrupt host cellular physiology and prevent bacterial clearance by host defense mechanisms. The specificity of these toxins for eukaryotic cells depends on the presence of substrate targets and eukaryotic cofactors responsible for effector activation. We used a combined biochemical and proteomic approach to identify Cu(2+), Zn(2+)-superoxide dismutase (SOD1) as a cofactor that activates the phospholipase activity of ExoU. Recombinant ExoU (rExoU) was activated in a dose-dependent manner by either bovine liver SOD1 or the yeast ortholog, Sod1p, but not by either Fe or Mn-containing SODs from E. coli or small molecule SOD mimetics. Inhibitor studies indicated that SOD enzymatic activity was not required for the activation of rExoU. The physical interaction between rExoU and SOD was demonstrated by capture techniques using either of the two proteins immobilized onto the solid phase. Identification of SOD as a cofactor allowed us to develop a new assay using a fluorescent substrate to measure the phospholipase activity of rExoU. The ability of SOD to act as a cytoplasmic cofactor stimulating ExoU phospholipase activity has significant implications for the biological activity of the toxin. Further elucidation of the structural mechanism of ExoU activation by this eukaryotic cofactor may provide a rational approach to the design of inhibitors that can diminish tissue damage during infection by ExoU-producing strains of P. aeruginosa.     

Properties of a cell growth inhibitor produced by mouse embryo fibroblasts

Secondary mouse embryo fibroblasts produce a growth inhibitor with the character of a thermolabile, nondialysable protein. The inhibitor was harvested from conditioned medium, and following G-75 Sephadex fractionation it was isolated in one peak which consisted of two fractions eluting at approximately two thirds of the bed volume of the column where approximately 80 percent of the original activity was recovered with an increase in specific activity of about tenfold. Polyacrylamide gradient gel electrophoresis of fractions from L-[35S] methionine-labelled conditioned medium showed that the two fractions with growth inhibitory activity contained some 4-5 bands and shared the two major components. Cell cycle studies showed that the growth inhibitory effect was exerted after addition during early and late G1 and during S phase, and morphological studies showed that where growth was inhibited the morphological expression of the cells was altered.     

Substrate-attached serum and cell proteins in adhesion of mouse fibroblasts.

The effects of serum and coatings of substrate-attached material (SAM, which remains tightly adherent to the substrate after EGTA-mediated removal of cells) on the kinetics of attachment of DNA-radiolabeled BALB/c 3T3. SV40-transformed 3T3, and concanavalin A-selected revertant cells to glass coverlips were studied. The presence of serum in the medium of attaching cells had a marked effect on (1) the initial time lag before stable attachment of cells, (2) the maximum level of attached cells, (3) the stability of attachment, and (4) pseudopodial spread of the cell over the substrate. These serum effects could be mimicked by measuring attachment in medium without serum and with use of serum-preadsorbed or 3T3 SAM-coated coverslips. Enzymatic treatment of serumpreadsorbed substrates indicated that the factor(s) in serum which affects attachment is very trypsin-sensitive. Serum preadsorption of substrates stimulated attachment of SVT2 cells in medium with serum in a manner very similar to the effects of 3T3 SAM coating, while attachment of 3T3 SAM coating, while attachment of 3T3 or revertant cells was unaffected. Slab gel electrophoretic analysis (PAGE-SDS gels) identified eight major serum proteins by Coomassie blue staining (a) which bind to the substrate in the absence of cells and (b) which persist on the substrate after growth to confluence of 3T3 or SVT2 cells; this suggests that major breakdown or serum-adsorbed components does not occur during growth of normal or transformed cells. Seven radioactive SAM proteins were detected by autoradiography in 3T3 or SVT2 SAM electropherograms -- two of which are high molecular weight components which correspond to the glucosamine-radiolabeled hyaluronate proteoglycans observed previously; the remaining five are newly-identified proteins in SAM (one of these proteins appears to be actin). 3T3 and SVT2 cells have unique proportions of these seven components. The data are consistent with the idea that normal or virus-transformed cells do not attach directly to the culture substrate, but to specific classes of substrate-adsorbed serum proteins via deposition of specific classes of cell surface proteins and polysaccharides.     

Mechanism of resistance to ricin toxin in selected mouse lymphoma cell lines

The role of impaired toxin uptake in conferring cellular resistance to the plant toxin RCA_II (ricin) has been examined using a murine BW5 147 lymphoma line and a toxin-resistant variant (BW5 147 Ric^R.3) selected by repeated exposure to RCA_II. The toxin-resistant variant is 250 times more resistant to RCA_II in long-term growth experiments and 1,000 times more resistant in short-term protein synthesis assays. Experiments with ferritin-conjugated ¹²⁵I-labeled RCA_II (ferritin-¹²⁵I-RCA_II) indicated that toxin binding to sensitive and resistant cells is similar at low toxin concentrations where maximum differential cytotoxicity occurs but that major difference exist with respect to toxin uptake. In sensitive cells toxin is internalized via endocytosis, and as seen previously in other systems subsequent rupture of some of the toxin-containing endocytotic vesicles releases toxin into the cytoplasm, where it inhibits protein synthesis. The process of toxin transfer to the cytoplasm is presumed to account for the one-hour lag before toxin-induced inhibition of protein synthesis can be detected. Endocytotic uptake of toxin is impaired in resistant BW5147Ric^R.3 cells, and they are unaffected by toxin concentrations that inhibit protein synthesis and kill sensitive parental cells. Killing of resistant cells at low toxin concentrations was accomplished by encapsulating RCA_II into lipid vesicles capable of fusing with the plasma membrane. Direct introduction of toxin into resistant cells using lipid vesicles as carriers produced rapid inhibition (< 15 min) of protein synthesis and eliminated the lag in toxin action seen in sensitive cells exposed to free toxin. These findings are discussed in relation to the mechanism of toxin action and proposals that toxin activity requires structural modification of the toxin molecule at the cell surface before transport into the cell.