A novel ganglioside, de-N-acetyl-GM3 (II3NeuNH2LacCer), acting as a strong promoter for epidermal growth factor receptor kinase and as a stimulator for cell growth

A novel ganglioside, de-N-acetyl-GM3 (neuraminyllactosylceramide, II3NeuNH2LacCer), was found in the monosialoganglioside fraction of A431 cells and B16 melanoma cells by high-performance liquid chromatography, thin-layer chromatography, and immunoblotting with its specific monoclonal antibody DH5. This novel type of membrane ganglioside strongly enhanced the kinase activity associated with the epidermal growth factor (EGF) receptor, and it showed 32, 35, and 12% growth stimulation as compared with control cultures of A431, Swiss 3T3, and B16 melanoma cells, respectively. Exogenously added de-N-acetyl-GM3 did not alter the affinity of EGF binding to its receptor. These properties of de-N-acetyl-GM3 are in striking contrast to those of GM3 and its lyso derivative (lyso-GM3) which were previously shown to inhibit EGF receptor kinase activity and to inhibit growth in the same cells. These data indicate that de-N-acetylation at the sialic acid moiety of GM3 ganglioside is an important mechanism for modulation of EGF-dependent cell growth. The mechanism is antagonistic to that of GM3-dependent modulation of receptor function.     

Evidence that type II insulin-like growth factor receptor is coupled to calcium gating system

In competent Balb/c 3T3 cells primed with epidermal growth factor (primed competent cells), insulin-like growth factor-II (IGF-II) stimulated calcium influx in a concentration dependent manner with the ED50 of 450 pM. When receptor-bound [125I]IGF-II was cross-linked by use of disuccinimidyl suberate, a 240 K-Da protein was radiolabeled. Excess amount of unlabeled IGF-II inhibited the affinity-labeling of the 240 K-Da protein. To further examine whether IGF-II stimulates calcium influx by acting on the type II IGF receptor, we employed polyclonal antibody raised against rat type II IGF receptor, R-II-PABl. This antibody immunoprecipitated the type II IGF receptor and inhibited IGF-II binding in Balb/c 3T3 cell membrane without affecting IGF-I binding. In primed competent cells, R-II-PABl elicited an agonistic action in stimulating [3H]thymidine incorporation. Under the same condition, R-II-PABl elicited a marked stimulation of calcium influx. These results suggest that, in Balb/c 3T3 cells, 1) relatively low concentrations of IGF-II act mainly on the type II IGF receptor; 2) the type II IGF receptor is coupled to a calcium gating system; and 3) binding of a ligand to the type II IGF receptor leads to the stimulation of DNA synthesis.     

Laminin synthesized by stationary and migrating rat liver epithelial cells lacks the A chain

The expression of laminin chains was analyzed in normal and Ha-ras1-transformed rat liver epithelial cells. The normal, nontumorigenic cells were induced to migrate by epidermal growth factor, whereas the Ha-ras1-transformed, malignant derivatives migrate constitutively. None of these cells express a typical (EHS-like) laminin A chain. Immunoprecipitation of [35S]-methionine-labeled liver cell lysates with an antibody against EHS-laminin revealed B1 and B2 chains and, in addition, two high Mr polypeptides. These polypeptides were not recognized by the antibody in immunoblots, suggesting that they might constitute alternative laminin A chains. Analysis of the expression of all three laminin chains at the RNA and protein level revealed that the pattern of expression of the stationary cells does not differ from that of the migratory ones and is also not influenced by epidermal growth factor. These results indicate that expression of a typical laminin A chain by rat liver epithelial cells is not required for the secretion and deposition of the protein in the extracellular matrix. The data also indicate that an EHS-like laminin A chain is not required for the migration of these epithelial cells.     

Epidermal growth factor induces the accumulation of calpactin II on the cell surface during membrane ruffling

Confluent and proliferatively quiescent T51B rat liver epithelial cells provide a cellular model for the study of epidermal growth factor (EGF) effects in non-neoplastic cells. Immunoreactive calpactin II, a well-known substrate for EGF-receptor kinase, was found predominantly in the cytosol, although a second immunoreactive pool was found in a Triton X-100-extractable membrane fraction. Stimulation with EGF resulted in a rapid and transient (2-5 min) formation of ruffles at the cell surface and at the cell-cell contacts. Both calpactin II and filamentous actin were found co-localized at the membrane ruffles. Immunoprecipitations of membrane-bound calpactin II from 32P-labeled cells indicate a transient EGF-dependent phosphorylation of calpactin II correlating with membrane ruffling. These results suggest a temporal (2-5 min) function for calpactin II at the plasma membrane during the EGF-induced mitogenesis of T51B cells.     

Calcium channels are present in the apical plasma membranes of the hepatopancreatic B-cells of<Emphasis Type="Italic"> Marsupenaeus japonicus</Emphasis>

This study demonstrates the existence of calcium channels in the apical membranes of the hepatopancreatic blister (B) cells of Marsupenaeus japonicus. Using brush-border membrane vesicles we demonstrated that the channel-mediated calcium passive flux was saturable and was stimulated by a transmembrane electrical potential difference and inhibited by barium. We raised a monoclonal antibody (Mab 24A4) against the calcium channel, which allowed us to inhibit the channel-mediated calcium uptake. By immunocytochemistry, using Mab 24A4, we demonstrated that these channels are located at the apical membrane of hepatopancreatic B cells. Finally, by measuring the calcium uptake in R- and B-enriched cell suspensions, we showed that only the plasma membrane of the B cells expresses a channel-mediated calcium uptake inhibited by barium, verapamil and the monoclonal antibody 24A4. The plasma membrane of R cells did not show calcium channels.Abbreviations ELISA enzyme-linked immunosorbent assay - BBMV brush-border membrane vesiclesCommunicated by G. Heldmaier     

Inhibition of murine leukemia (WEHI-3B and L1210) proliferation by cholera toxin B subunit

Cholera holotoxin produces both stimulation and inhibition of the growth of different cell populations. These opposite effects were both attributed to the enzymatic activity of the subunit A that activates adenylate cyclase, increasing the intracellular level of cAMP. We observed that the B subunit of cholera toxin produced by itself an inhibition of the 'in vitro' growth of two murine leukemia cell lines (L1210 limphoid leukemia and WEHI-3B myelomonocytic leukemia). The sensitivity of WEHI-3B cells towards cholera toxin was about 5000-times higher than that of the L1210 cells, whereas the two leukemias showed an identical sensitivity to the B subunit (IC50 = 5.10(-10) M for L1210 and 10(-10) M for WEHI-3B). The inhibition produced by the B subunit was neutralized by GM1 and in a minor degree by type II gangliosides. The two leukemias showed a remarkable difference in their gangliosides contents (L1210 cells contained GM1 (80.6%) and GM2 (19.4%), while WEHI-3B cells contained GM1 (28.2%), Fuc-GM1 (44.9%) and a band (26.9%) with a chromatographic mobility between GD1a and GD1b). The inhibition could be explained by a competitive mechanism between the B subunit and some autocrine factor binding GM1-containing receptors. Our data strengthen the suggestion to consider gangliosides as very important pleiotropic biomodulators.     

Insulin-like growth factors, insulin, and epidermal growth factor cause rapid cytoskeletal reorganization in KB cells. Clarification of the roles of type I insulin-like growth factor receptors and insulin receptors

Insulin-like growth factor (IGF) I (greater than or equal to 10(-10)M, insulin-like growth factor II (greater than or equal to 10(-9) M), insulin (greater than or equal to 10(-9) M, and epidermal growth factor (EGF, greater than or equal to 10(-11) M) caused rapid membrane ruffling in KB cells. The morphological change was observed within 1 min after the addition of these growth factors and was accompanied by microfilament reorganization, but not by microtubule reorganization. IGF-I, IGF-II, and insulin induced morphologically very similar or identical membrane ruffles with the order of potency IGF-I greater than IGF-II greater than insulin, whereas EGF-induced membrane ruffles were morphologically different. KB cells possessed EGF receptors, type I IGF receptors, and insulin receptors, but few or no type II IGF receptors. Monoclonal antibody against type I IGF receptors, which completely inhibited the binding of 125I-IGF-I to the cells but did not inhibit the binding of 125I-insulin, caused marked inhibition of IGF-I (10(-8) M)-stimulated membrane ruffling. IGF-II (10(-8) M)-stimulated membrane ruffling was partially inhibited in the presence of this antibody, but insulin (10(-7) M)-stimulated membrane ruffling was only slightly inhibited. In contrast, monoclonal antibody against insulin receptors blocked insulin (10(-7) M) stimulation, but not IGF-I (10(-8) M) stimulation, of membrane ruffling. Thus, this study provides evidence that IGF-I and insulin act mostly through their own (homologous) receptors and that IGF-II acts by cross-reacting with both type I IGF and insulin (heterologous) receptors in causing rapid alterations in cytoskeletal structure.     

SCP-2/SCP-x gene ablation alters lipid raft domains in primary cultured mouse hepatocytes

Although reverse cholesterol transport from peripheral cell types is mediated through plasma membrane microdomains termed lipid rafts, almost nothing is known regarding the existence, protein/lipid composition, or structure of these putative domains in liver hepatocytes, cells responsible for the net removal of cholesterol from the body. Lipid rafts purified from hepatocyte plasma membranes by a nondetergent affinity chromatography method were: i) present at 33 +/- 3% of total plasma membrane protein; ii) enriched in key proteins of the reverse cholesterol pathway [scavenger receptor class B type I (SR-B1), ABCA1, P-glycoprotein (P-gp), sterol carrier protein-2 (SCP-2)]; iii) devoid of caveolin-1; iv) enriched in cholesterol, sphingomyelin, GM1, and phospholipids low in polyunsaturated fatty acid and double bond index; and v) exhibited an intermediate liquid-ordered lipid phase with significant transbilayer fluidity gradient. Ablation of the gene encoding SCP-2 significantly altered lipid rafts to: i) increase the proportion of lipid rafts present, thereby increasing raft total content of ABCA1, P-gp, and SR-B1; ii) increase total phospholipids while decreasing GM1 in lipid rafts; iii) decrease the fluidity of lipid rafts, consistent with the increased intermediate liquid-ordered phase; and iv) abolish the lipid raft transbilayer fluidity gradient. Thus, despite the absence of caveolin-1 in liver hepatocytes, lipid rafts represented nearly one-third of the mouse hepatocyte plasma membrane proteins and displayed unique protein, lipid, and biophysical properties that were differentially regulated by SCP-2 expression.     

Activation of bradykinin B2 receptors increases calcium entry and intracellular mobilization in C9 liver cells.

In C9 rat liver cells bradykinin and kallidin increased (approximately 2-fold) the intracellular concentration of calcium, but the B1 agonist, des-Arg9-bradykinin did not. The effect of bradykinin was inhibited by the B2 antagonists, Hoe 140 and N-alpha-adamantaneacetyl-D-Arg-[Hyp3, Thi5,8, D-Phe7]-bradykinin, but not by the B1 antagonist, des-Arg9-[Leu8]-bradykinin. The action of bradykinin was diminished, but not abolished, in medium without calcium. The peptide was able to increase intracellular calcium concentration in cells treated with thapsigargin. Bradykinin action was not observed in cells previously stimulated with this local mediator: however, under the same conditions, angiotensin II induced a clear increase in intracellular calcium concentration. Our data indicate that activation of bradykinin B2 receptors increase intracellular calcium concentrations by inducing both gating of the cation and intracellular mobilization in C9 liver cells. In addition, homologous desensitization was observed.     

Cyclic AMP-independent effects of cholera toxin on B cell activation. II. Binding of ganglioside GM1 induces B cell activation.

Although the physiologic function of gangliosides is unknown, evidence suggests they play a role in the regulation of cell growth. The binding of ganglioside GM1 by recombinant B subunit of cholera toxin (rCT-B) inhibited mitogen-stimulated B cell proliferation without elevating intracellular cAMP. CT-B paradoxically enhanced the expression of MHC class II (Ia) molecules and minor lymphocyte-stimulating determinants without altering the expression of some other immunologically relevant B cell surface Ag. Increased expression of Ia was not detected until 4 h after stimulation, kinetics similar to those seen when B cells are stimulated with anti-Ig antibody or IL-4, suggesting that the enhancement was not the result of redistribution of existing cell surface markers but rather the result of a new metabolic event. Both the inhibitory and stimulatory effects of CT-B could be blocked by incubation of CT-B with ganglioside GM1. Furthermore, enhancement of the CT-B-mediated effect was seen when additional ganglioside GM1 was incorporated into the B cell membrane. rCT-B with a mutation that interfered with its binding to ganglioside GM1 did not enhance Ia expression. Taken together, these results indicate that the observed effects of CT-B were most likely mediated through the binding of cell surface ganglioside GM1. CT-B-mediated stimulation of Ia expression provides a potential explanation for the previously described ability of CT-B to act as an immunoadjuvant. These results suggest that the binding of ganglioside GM1 has multiple B cell growth-regulating effects.     

Hexosaminidase isozyme in type O Gm2 gangliosidosis (Sandhoff-Jatzkewitz disease).

The residual enzyme of the fibroblasts of a child with homozygous type 0 GM2 gangliosidosis (Sandhoff-Jatzkewitz disease) has been found to correspond with a minor fraction of enzyme which can be isolated from normal fibroblasts by repeated chromatography. This enzyme is designated as hexosaminidase (hex) S. It reacts with antiserum prepared against homogeneous hex A but not with serum prepared against homogeneous hex B. These findings support our previously described model of the relationship between hex A and hex G: hex A has the structure (alpha beta)3, while hex B is (beta)6. Type B GM2 gangliosidosis (Tay-Sachs disease) is the alpha- mutation, while type 0 GM2 gangliosidosis (Sandhoff-Jatzkewitz disease) is the beta- mutation. In the absence of normal beta subunits there is increased polymerization of alpha subunits forming hex S, which probably has a structure of (alpha)6. A parallel between the thalassemias and GM2 gangliosidosis is evident: deficiency of one of the chains of which the protein is composed leads to an excess of polymers comprised of the other chains. In type B GM2 gangliosidosis, the excess of beta chanis leads to increased amounts of hex B beta)6; in type 0 GM2 gangliosidosis, the excess of alpha chains leads to formation of increased amounts of the alpha chain polymer, hex S.     

Calcium channels in PDGF-stimulated A172 cells open after intracellular calcium release and are not voltage-dependent.

Using laser image cytometry and Indo-1 fluorescence, we investigated the intracellular free Ca2+ concentration ([Ca2+]i) of confluent A172 human glioblastoma cells stimulated by the BB homodimer of platelet-derived growth factor (PDGF-BB). The shape of the calcium transients and the delay time between stimulation and the beginning of the transient varied considerably. The percentage of responsive cells, the peak [Ca2+]i and the duration of the response were directly related to PDGF-BB dose, while the delay time was inversely related; the maximal response occurred at a PDGF-BB concentration of 20 ng/ml. Studies with EGTA and inorganic calcium-channel blockers (Ni2+, La3+) showed that the increase of [Ca2+]i resulted from initial release of intracellular stores and subsequent calcium influx across the plasma membrane. Opening of calcium channels in the plasma membrane, monitored directly by studying Mn2+ quenching of Indo-1 fluorescence, was stimulated by PDGF-BB and blocked by La3+; the opening occurred 55 +/- 10 s after the initial increase in [Ca2+]i. Therefore, in these tumor cells, intracellular release always occurs before channel opening in the plasma membrane. Depolarization of cells with high extracellular [K+] did not generally induce calcium transients but did decrease calcium influx. L-type calcium-channel blockers (verapamil, nifedipine, and diltiazem) had little or no effect on the calcium influx induced by PDGF-BB. These results indicate that PDGF-BB induces calcium influx by a mechanism independent of voltage-sensitive calcium channels in A172 human glioblastoma cells.     

Induction of proliferation by high molecular weight B cell growth factor or low molecular weight B cell growth factor is associated with increases in intracellular calcium in different subpopulations of human B lymphocytes

The activation of resting B cells with anti-surface Ig is associated with transient increases in intracellular calcium. In the present study, we demonstrate that stimulation of B cells which have already been activated by Staphylococcus aureus Cowan I (Sac), with high molecular weight B cell growth factor (HMW-BCGF) or low molecular weight B cell growth factor (LMW-BCGF), but not IL-2, IL-4, or interferon-gamma, is associated with an increase in intracellular calcium, which is modest compared to that seen with anti-Ig (approximately 100 nM vs approximately 400 nM). The increases in intracellular calcium induced by HMW-BCGF or LMW-BCGF occur in distinct but overlapping subpopulations of B cells. Thus, increases in intracellular calcium in human B cells occur not only upon activation but also upon the induction of proliferation by certain (but not all) B cell growth factors. Presumably, the effect of increasing intracellular calcium during the induction of proliferation is to modify a different group of intracellular molecules than those induced during activation.     

Stable Transfection of GM1 Synthase Gene into GM1-Deficient NG108-15 Cells,CR-72 Cells,Rescues the Responsiveness of TRK-Neurotrophin Receptor to Its Ligand,NGF

Previous studies from this laboratory and others have suggested the evidences that acidic glycosphingolipid, ganglioside GM1 (GM1), is an endogenous regulator of high affinity nerve growth factor receptor, Trk, which is an essential factor for the normal development and differentiation of neuronal cells by forming a complex with Trk. The present study was aimed to examine whether Trk expressed in cells that are deficient in endogenous GM1 due to the mutation of GM1 synthase gene (NG-CR72 cells) is responsive to its ligand nerve growth factor and how genetic restoration of GM1 synthase gene by a stable transfection of the gene affects the function of the Trk protein. The data clearly showed that (1) confocal lazor microscopic studies disclosed NG-CR72 cells are really deficient in GM1, (2) stable transfection of GM1 synthase cDNA into these cells (NG-CR72G cells) restores the expression of GM1 in the cells, and (3) Trk protein is expressed in NG-CR72 cells but its location seemed not to be on the plasma membrane, whereas we clearly observed that the Trk protein is expressed on the plasma membrane in NG-CR72G cells. (4) NGF did not elicit the autophosphorylation of the Trk protein in GM1 deficient NG-CR72 cells but did elicit the activation of the Trk protein in NG-CR72G cells with an activation of mitogen activated protein kinase. These studies strongly suggested that GM1 is necessary for the normal expression of the Trk protein function and for normal targeting of the Trk protein to the plasma membrane.     

Monoclonal antibody DH12 reacts with a cell surface and a precursor form of the beta subunit of the human fibronectin receptor

Monoclonal and polyclonal antibodies were raised against a placenta plasma membrane protein preparation, which was obtained by fractionation on Blue B dye matrix and by HPLC-anionexchange, and which was shown to contain fibronectin receptors. Immunochemical and functional evidence showed that monoclonal antibody DH12 recognized the beta subunit of the human fibronectin receptor on fibroblasts. This monoclonal antibody reacted with two proteins in Western blots and in double immune precipitations of whole cell preparations. Only the higher Mr protein became labeled by surface iodination of intact fibroblasts. The lower Mr protein is thought to be an intracellular precursor of the beta subunit of the fibronectin receptor.     

Studies on a PMCA-like protein in the outer mantle epithelium of <Emphasis Type="Italic">Anodonta cygnea</Emphasis>: insights on calcium transcellular dynamics

Early studies on the outer mantle epithelium (OME) cells of the freshwater bivalve Anodonta cygnea (Linnaeus, 1758) revealed high ionic calcium concentrations by electrophysiological methods and subsequently a high tendency to reach an intracellular toxic condition. This toxicity could be neutralized by specific mechanisms in the cytosol of OME cells of A. cygnea. The present immunocytochemistry studies of OME cells by light and transmission electron microscopy (TEM) clearly showed a positive reaction of an antibody directed against the human plasma membrane Ca²⁺-ATPase 1 (PMCA-1) in the cytoplasm of OME cells. Also, western blot analysis of different fractions of OME cells with anti human PMCA-1 and C28R2 antibodies confirmed the presence of a PMCA-like protein with an unusual topographical localization and a molecular weight of only 70–80 kDa. These results lead us to speculate that this PMCA-like protein is distributed either in the plasma membrane or in the entire cytosol, where it eventually regulates intracellular calcium levels. Interestingly, the antibody reactions showed seasonal variations, being highest in OME samples prepared during summer when A. cygnea live under natural acidosis and absent in samples taken in winter conditions, which is in accordance with the seasonal variation of shell calcification rates. During winter, PMCA-1 antibody reaction was also detected in OME cells of animals kept only under experimentally induced acidosis conditions. Therefore, we assume that a functional role for this PMCA-like protein in the intracellular calcium regulation of OME cells during the mineralization of the shells of A. cygnea can be speculated.     

Immunological discrimination of intralysosomal, cytosolic, and two membrane sialidases present in rat tissues

Cytosolic sialidase was purified from rat skeletal muscle, and the purified enzyme migrated as a single band of Mr 43,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A polyclonal antibody raised against the enzyme inhibited and immunoprecipitated rat liver cytosolic sialidase as well as the muscle enzyme but failed to cross-react with the intralysosomal sialidase of rat liver and membrane sialidases I (synaptosomal) and II (lysosomal) of rat brain. The antibody against brain membrane sialidase I (anti-I) and that against sialidase II (anti-II), which could be useful to discriminate the two enzymes, did not cross-react with the intralysosomal and cytosolic sialidases of liver. Although more than 90% of liver plasma membrane sialidase was immunoprecipitated with anti-I, only 60% of liver lysosomal membrane sialidase was immunoprecipitated with anti-II, the remainder being immunoprecipitated with anti-I. In confirmation of these data, liver lysosomal membrane exhibited two peaks of ganglioside sialidase corresponding to the membrane sialidases I and II on Aminohexyl-Sepharose chromatography while only one peak of ganglioside sialidase corresponding to sialidase I was observed for liver plasma membrane. These results indicate that the four types of rat sialidase are proteins distinct from one another and that the three kinds of antisera described above are useful for discriminating these sialidases qualitatively and probably quantitatively.     

Endogenous GM1 Ganglioside of the Plasma Membrane Promotes Neuritogenesis by Two Mechanisms

The influence of GM1 on the neuritogenic phase of neuronal differentiation has been highlighted in recent reports showing upregulation of this ganglioside in the plasma and nuclear membranes concomitant with axonogenesis. These changes are accompanied by alterations in Ca²⁺ flux which constitute an essential component of the signaling mechanism for axon outgrowth. This study examines 2 distinct mechanisms of induced neurite outgrowth involving plasma membrane GM1, as expressed in 3 neuroblastoma cell lines. Growth of Neuro-2a and NG108-15 cells in the presence of neuraminidase (N'ase), an enzyme that increases the cell surface content of GM1, caused prolific outgrowth of neurites which, in the case of Neuro-2a, could be blocked by the B subunit of cholera toxin (Ctx B) which binds specifically to GM1; however, the latter agent applied to NG108-15 cells proved neuritogenic and potentiated the effect of N'ase. With N18 cells, the combination was also neuritogenic as was Ctx B alone, whereas N'ase by itself had no effect. Neurite outgrowth correlated with influx of extracellular Ca²⁺, determined with fura-2. Treatment of NG108-15 and N18 cells with Ctx B alone caused modest but persistent elevation of intracellular Ca²⁺ while a more pronounced increase occurred with the combination Ctx B + N'ase. Treatment with N'ase alone also caused modest but prolonged elevation of intracellular Ca²⁺ in NG108-15 and Neuro-2a but not N18; in the case of Neuro-2a this effect was blocked by Ctx B. Neuro-2a and N18 thus possess 2 distinctly different mechanisms for neuritogenesis based on Ca²⁺ modulation by plasma membrane GM1, while NG108-15 cells show both capabilities. The neurites stimulated by N'ase + Ctx B treatment of N18 cells were shown to have axonal character, as previously demonstrated for NG108-15 cells stimulated in this manner and for Neuro-2a cells stimulated by N'ase alone.