Membrane gangliosides modulate interleukin-2-stimulated T-lymphocyte proliferation.

Membrane gangliosides appear to modulate signal transduction by several growth factor receptors. We have investigated the possible regulation of IL-2-induced proliferation signals by gangliosides. Low concentrations of cholera toxin B subunit (CT-B), which binds specifically to GM1 ganglioside, greatly inhibited IL-2-stimulated DNA synthesis in the IL-2-dependent cell line CTLL-2, but had no effect on proliferation of HT-2. GM1 levels proved to be very low in HT-2 compared to CTLL-2. Large increases in membrane-associated GM1 could be achieved in both cell lines by incubation with exogenous GM1, resulting in a high degree of inhibition of proliferation by CT-B for both CTLL-2 and HT-2. Inhibition was blocked by large unilamellar vesicles containing GM1, but not by vesicles of lipid alone. The time course of CT-B inhibition for CTLL-2 synchronized in G0-G1, indicated that the negative growth signal acts relatively early in the IL-2 activation pathway. CT-B did not affect binding of IL-2 to high-affinity IL-2r. The inhibitory effects of CT-B could not be reversed by pertussis toxin, suggesting that a G protein is probably not involved. These results show that CT-B binding to either endogenous or inserted GM1 can modulate IL-2-induced lymphocyte proliferation.     

CIS associates with the interleukin-2 receptor beta chain and inhibits interleukin-2-dependent signaling.

CIS is a cytokine-induced SH2-containing protein that was originally cloned as an interleukin (IL)-3-inducible gene. CIS is known to associate with the IL-3 receptor beta chain and erythropoietin receptor and to inhibit signaling mediated by IL-3 and erythropoietin. We now demonstrate that CIS also interacts with the IL-2 receptor beta chain (IL-2Rbeta). This interaction requires the A region of IL-2Rbeta (residues 313-382), which also mediates the association of IL-2Rbeta with Lck and Jak3. Correspondingly, CIS inhibits functions associated with both of these kinases: Lck-mediated phosphorylation of IL-2Rbeta and IL-2-mediated activation of Stat5. Thus, we demonstrate that CIS can negatively control at least two independent IL-2 signaling pathways. Although a functional SH2 binding domain of CIS was not required for its interaction with IL-2Rbeta in vitro, its phosphotyrosine binding capability was essential for the inhibitory action of CIS. On this basis, we have generated a mutant form of CIS protein with an altered SH2 domain that acts as a dominant negative and should prove useful in further understanding CIS action.     

Role of insulin receptor substrate-2 in interleukin-9-dependent proliferation

Interleukin-9 (IL-9) stimulation results in JAK, STAT and IRS1/2 phosphorylation. The role of IRS adaptor proteins in IL-9 signaling is not clear. We show that IL-9 induces IRS2 phosphorylation and association with phosphatidylinositol-3 kinase (PI 3-K) p85 subunit in TS1 cells and BaF/9R cells, which proliferate upon IL-9 stimulation. We observed a PI 3-K-dependent phosphorylation of protein kinase B (PKB) in TS1 cells, but not in BaF/9R, nor in other IL-9-dependent cell lines. Finally, 32D cells that were transfected with the IL-9 receptor but lack IRS expression survived in the presence of IL-9. Ectopic IRS1 expression allowed for IL-9-induced proliferation, in the absence of significant PKB phosphorylation.     

2-Mercaptoethanol acts as a potentiating factor of interleukin-2-dependent lymphocyte proliferation

An interleukin-2 (IL-2) dependent cell line which could be grown continuously with crude concanavalin A-stimulated rat or mouse spleen cell culture supernatant could not survive for over 48 hr in the culture medium supplemented with partially purified or recombinant IL-2. The cell growth was restored by adding another factor obtained from the same crude culture supernatant. This potentiating activity which was physicochemically inseparable from serum albumin was also obtained from the culture medium containing 2-mercaptoethanol (2-ME) and incubated at 37 C for 24 hr without the spleen cells. By further experiments, it was demonstrated that 2-ME itself had the potentiating activity on the IL-2-dependent proliferation of this cell line and cysteine mediated the activity of 2-ME. The cells could not enter S-phase in the absence of 2-ME even in the presence of IL-2.     

Polyamine biosynthesis is necessary for interleukin-2-dependent proliferation but not for interleukin-2 production or high-affinity interleukin-2 receptor expression

DL-alpha-Difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase [EC 4.1.1.17] (ODC), inhibited concanavalin A-induced proliferation of splenic mononuclear cells (SMNC). The inhibition was not reversed by interleukin-2 (IL-2) addition. Although DFMO did not affect the production of IL-2 or the expression of high-affinity IL-2 receptor, IL-2-dependent proliferation of SMNC was inhibited by DFMO, and the inhibition was reversed by exogenous putrescine. The inhibition of IL-2-dependent DNA synthesis appeared to be related to the decrease in intracellular polyamines. When the proliferation of SMNC was induced by IL-2, ODC activity was also increased. A similar result was obtained in the proliferation of an IL-2-dependent T cell line, CTLL. The time course of ODC induction was similar to that of IL-2 production by concanavalin A-stimulated SMNC. These results indicate that polyamine biosynthesis is necessary for IL-2-dependent proliferation, but not for IL-2 production or IL-2 receptor expression.     

Effect of micellar and bilayer gangliosides on proliferation of interleukin-2-dependent lymphocytes

Micellar gangliosides are potent inhibitors of the proliferation of the murine interleukin-2-dependent cell lines HT-2 and CTLL-2 in vitro. The glycolipids abolished both DNA and protein synthesis, and depressed cellular expansion, without affecting viability. These effects were reversible for at least 12 hr following ganglioside treatment. Highly sialylated gangliosides were more inhibitory, while structurally related molecules, including ganglioside oligosaccharides, simple and complex neutral glycosphingolipids, sulfatides, sphingomyelin, ceramides, and sphingosine had only small suppressive effects. Gangliosides were most effective as inhibitors when added during the first 4 hr of culture with the growth factor. Inhibition of DNA synthesis by gangliosides could be partially reversed by high concentrations of exogenous interleukin-2. Gangliosides incorporated into lipid bilayers, both multilamellar liposomes and unilamellar vesicles, were also effective inhibitors of interleukin-2-induced proliferation. Competition studies showed that both ganglioside micelles and lipid vesicles containing gangliosides prevented binding of 125I-interleukin-2 to high-affinity receptors on the lymphocyte surface. We have recently shown that gangliosides, in both micelles and lipid bilayer vesicles, are able to bind interleukin-2 (J. W. K. Chu and F. J. Sharom, Biochim, Biophys. Acta 1028, 205, 1990). Taken together, these results strongly suggest that inhibition of lymphocyte proliferation by gangliosides in micelles and vesicles arises as a direct result of competition between the glycolipids and high-affinity receptors for available interleukin-2.     

Mycophenolic acid inhibits IL-2-dependent T cell proliferation,but not IL-2-dependent survival and sensitization to apoptosis

Mycophenolic acid (MPA), the active metabolite of the immunosuppressive drug mycophenolate mofetil, is a selective inhibitor of inosine 5'-monophosphate dehydrogenase type II, a de novo purine nucleotide synthesis enzyme expressed in T and B lymphocytes and up-regulated upon cell activation. In this study, we report that the blockade of guanosine nucleotide synthesis by MPA inhibits mitogen-induced proliferation of PBL, an effect fully reversed by addition of guanosine and shared with mizoribine, another inhibitor of inosine 5'-monophosphate dehydrogenase. Because MPA does not inhibit early TCR-mediated activation events, such as CD25 expression and IL-2 synthesis, we investigated how it interferes with cytokine-dependent proliferation and survival. In activated lymphoblasts that are dependent on IL-2 or IL-15 for their proliferation, MPA does not impair signaling events such as of the extracellular signal-regulated kinase 2 and Stat5 phosphorylation, but inhibits down-regulation of the cyclin-dependent kinase inhibitor p27(Kip1). Therefore, in activated lymphoblasts, MPA specifically interferes with cytokine-dependent signals that control cell cycle and blocks activated T cells in the mid-G(1) phase of the cell cycle. Although it blocks IL-2-mediated proliferation, MPA does not inhibit cell survival and Bcl-x(L) up-regulation by IL-2 or other cytokines whose receptors share the common gamma-chain (CD132). Finally, MPA does not interfere with IL-2-dependent acquisition of susceptibility to CD95-mediated apoptosis and degradation of cellular FLIP. Therefore, MPA has unique functional properties not shared by other immunosuppressive drugs interfering with IL-2R signaling events such as rapamycin and CD25 mAbs.     

IL-4 inhibits IL-2 receptor expression and IL-2-dependent proliferation of human T cells

Recent studies have shown that IL-4 can affect lymphocyte responses to IL-2. To evaluate the effects of IL-4 on T cell responses to physiologically relevant stimuli, we studied normal human T cells cultured with a low concentration of anti-CD3 mAb and IL-2 in the presence and absence of added IL-4. The addition of IL-4 to cultures of T cells stimulated with anti-CD3 mAb and IL-2 reduced the proliferative response by 49 to 59%. The inhibitory effect was observed in 3-, 5-, and 7-day cultures. Inhibition was dose-dependent with maximal inhibition at concentrations greater than or equal to 5 to 10 U/ml IL-4. IL-4-mediated inhibition occurred early during the T cell response, inasmuch as addition of IL-4 after stimulation for 24 h did not result in significant inhibition. Phenotypic analyses of cells cultured in the presence of anti-CD3 mAb, IL-2, and IL-4 suggested that the mechanism of regulation by IL-4 involves the inhibition of IL-2R expression. The proportion of both CD4+ and CD8+ cells that expressed IL-2R in response to IL-2 was diminished in the presence of IL-4, although HLA-DR levels were unaffected. Soluble IL-2R was also reduced in supernatants of cultures stimulated with anti-CD3 mAb, IL-2, and IL-4 as compared to cultures stimulated with anti-CD3 mAb and IL-2. These findings indicate that when normal human T cells are stimulated in vitro in a manner that approximates a physiologic interaction with Ag in vivo, rIL-4 provides a potent inhibitory signal to IL-2 responsive cells that is likely mediated by IL-4-induced inhibition of IL-2R expression.     

Tazarotene-induced gene 1 interacts with Polo-like kinase 2 and inhibits cell proliferation in HCT116 colorectal cancer cells

Tazarotene-induced gene 1 (TIG1) is considered to be a tumor suppressor gene that is highly expressed in normal or well-differentiated colon tissues, while downregulation of TIG1 expression occurs in poorly differentiated colorectal cancer (CRC) tissues. However, it is still unclear how TIG1 regulates the tumorigenesis of CRC. Polo-like kinases (Plks) are believed to play an important role in regulating the cell cycle. The performance of PLK2 in CRC is negatively correlated with the differentiation status of CRC tissues. Here, we found that PLK2 can induce the growth of CRC cells and that TIG1 can prevent PLK2 from promoting the proliferation of CRC cells. We also found that the expression of PLK2 in CRC cells was associated with low levels of Fbxw7 protein and increased expression of cyclin E1. When TIG1 was coexpressed with PLK2, the changes in Fbxw7/cyclin E1 levels induced by PLK2 were reversed. In contrast, silencing TIG1 promoted the proliferation of CRC, and when PLK2 was also silenced, the proliferation of CRC cells induced by TIG1 silencing was significantly inhibited. The above research results suggest that TIG1 can regulate the tumorigenesis of CRC by regulating the activity of PLK2.     

Staphylococcus aureus Staphopain A inhibits CXCR2-dependent neutrophil activation and chemotaxis

The CXC chemokine receptor 2 (CXCR2) on neutrophils, which recognizes chemokines produced at the site of infection, plays an important role in antimicrobial host defenses such as neutrophil activation and chemotaxis. Staphylococcus aureus is a successful human pathogen secreting a number of proteolytic enzymes, but their influence on the host immune system is not well understood. Here, we identify the cysteine protease Staphopain A as a chemokine receptor blocker. Neutrophils treated with Staphopain A are unresponsive to activation by all unique CXCR2 chemokines due to cleavage of the N-terminal domain, which can be neutralized by specific protease inhibitors. Moreover, Staphopain A inhibits neutrophil migration towards CXCR2 chemokines. By comparing a methicillin-resistant S. aureus (MRSA) strain with an isogenic Staphopain A mutant, we demonstrate that Staphopain A is the only secreted protease with activity towards CXCR2. Although the inability to cleave murine CXCR2 limits in-vivo studies, our data indicate that Staphopain A is an important immunomodulatory protein that blocks neutrophil recruitment by specific cleavage of the N-terminal domain of human CXCR2.     

Cyld inhibits tumor cell proliferation by blocking Bcl-3-dependent NF-kappaB signaling

Mutations in the CYLD gene cause tumors of hair-follicle keratinocytes. The CYLD gene encodes a deubiquitinase that removes lysine 63-linked ubiquitin chains from TRAF2 and inhibits p65/p50 NF-kappaB activation. Here we show that mice lacking Cyld are highly susceptible to chemically induced skin tumors. Cyld-/- tumors and keratinocytes treated with 12-O-tetradecanoylphorbol-13 acetate (TPA) or UV light are hyperproliferative and have elevated cyclin D1 levels. The cyclin D1 elevation is caused not by increased p65/p50 action but rather by increased nuclear activity of Bcl-3-associated NF-kappaB p50 and p52. In Cyld+/+ keratinocytes, TPA or UV light triggers the translocation of Cyld from the cytoplasm to the perinuclear region, where Cyld binds and deubiquitinates Bcl-3, thereby preventing nuclear accumulation of Bcl-3 and p50/Bcl-3- or p52/Bcl-3-dependent proliferation. These data indicate that, depending on the external signals, Cyld can negatively regulate different NF-kappaB pathways; inactivation of TRAF2 controls survival and inflammation, while inhibition of Bcl-3 controls proliferation and tumor growth.     

Esterase D interacts with metallothionein 2A and inhibits the migration of A549 lung cancer cells in vitro

Esterase D (ESD) is a nonspecific esterase widely distributed in various organisms. ESD plays an important role in regulating cholesterol efflux, inhibiting viral replication and lung cancer growth. MT2A (metallothionein 2A) is the most important isoform of metallothionein (MTs) in human and high expression of MT2A in tumors represents poor prognosis and metastatic behavior. However, there are no reports about the molecular mechanism of ESD in the regulation of tumor metastasis. In this study, we found for the first time that activation ESD promoted its interaction with MT2A and decreased the protein level of MT2A, which resulting in the concentration of free zinc ions up-regulated, and inhibited the migration of A549 lung cancer cells in vitro.     

Physarum myosin light chain interacts with actin in a Ca2+-dependent manner

Actin-modulating activity was analysed with the 16,131-dalton calcium-binding light chain (CaLc, Kobayashi et al. (1988) J. Biol. Chem. 263, 305-313) of Physarum myosin, which is under an inhibitory Ca-control (Kohama and Kendrick-Jones (1986) J. Biochem. 99, 1433-1446). When skeletal muscle actin was polymerized in the presence of CaLc and Ca2+, increases in both viscosity and birefringence were reduced under high shear conditions. However, CaLc did not inhibit actin polymerization under no or low shearing forces, which was demonstrated by a variety of methods including fluorescence intensity measurements using pyrenyl actin. We propose that actin polymerized in the presence of CaLc and Ca2+ is easily fragmented under high shearing forces to produce the changes in viscosity and birefringence.     

Arachidonic acid-containing phosphatidylcholine inhibits lymphocyte proliferation and decreases interleukin-2 and interferon-gamma production from concanavalin A-stimulated rat lymphocytes

The proliferation of concanavalin A (Con A)-stimulated rat lymphocytes was markedly inhibited by phosphatidylcholine containing arachidonic and stearic acids (PC(A-S)), but not by phosphatidylcholine containing oleic and stearic acids or phosphatidylinositol containing arachidonic and stearic acids. The concentration of PC(A-S) which inhibited Con A-stimulated proliferation by 50% was 31 microM and near total inhibition was observed at 154 microM . Phosphatidylserine containing only oleic acid enhanced proliferation by 37% at a concentration of 31 microM , but phosphatidylethanolamine and phosphatidylcholine containing only oleic acid did not affect proliferation at this concentration. It is concluded that both the head group and the fatty acid composition contribute to the influence of phospholipids on lymphocyte proliferation. The effects of PC(A-S) on T-lymphocyte responses were investigated further. In parallel with the inhibition of proliferation PC(A-S) caused a concentration-dependent decrease in the production of the Th1-type cytokines interleukin (IL)-2 and interferon (IFN)-gamma; inhibition of cytokine production was >85% at the highest concentration of PC(A-S) used (154 microM ). Production of the Th2-type cytokines IL-4 and IL-10 was not affected. The possible role of prostaglandins in mediating the effects of PC(A-S) was examined by adding indomethacin into the medium and the participation of lipid peroxidation was examined by adding vitamin E and vitamin C. Indomethacin and vitamin E did not affect the inhibition caused by PC(A-S) but vitamin C caused a partial reversal. It is concluded that inhibition of T-lymphocyte proliferation by phospholipids involves both the head group and the fatty acyl chains, that this inhibition is not mediated by prostaglandins but may involve some form of oxidant stress and that some phospholipids (e.g., PC(A-S)) can markedly influence cytokine profiles.     

Mechanism of cyclosporin A-induced immunosuppression. Cyclosporin A inhibits receptor-mediated and non-receptor-mediated lymphokine production as well as interleukin-2-induced proliferation in cloned T lymphocytes

The effects of cyclosporin A (CyA) on the activation processes of cloned murine cytotoxic T lymphocytes (CTL) have been examined. With the use of Day 7 resting cloned CTL it was possible to separate the functions of lymphokine production (macrophage-activating factor, MAF) and interleukin 2 (IL-2)-induced proliferation of these cells. The effect of CyA on each of these activities was analyzed independently. CyA was found to inhibit both receptor-mediated MAF production in response to stimulation with antigen and lectin and MAF production in response to non-receptor-mediated stimulation (by anti-Thy-1 antibodies, ionophore, and phorbol ester). Further, CyA was observed to inhibit the re-entry of these resting CTL into the cell cycle upon stimulation with IL-2. The effect of CyA on MAF production did not appear to be due to inhibition of the signal-transducing mechanism involved in this process (i.e., inositol lipid hydrolysis, calcium mobilization, and protein phosphorylation). The action of CyA on the IL-2-induced proliferation was not due to inhibition of IL-2 receptor expression or the binding of IL-2 to its receptor. Thus, CyA appeared to mediate its suppressive effects on MAF production and IL-2-induced proliferation through an action on some later step(s) in the signal pathways of these activities.     

GM1 specifically interacts with alpha-synuclein and inhibits fibrillation

The aggregation of alpha-synuclein is believed to be a key step in the etiology of Parkinson's disease. Alpha-synuclein is found in the cytosol and is associated with membranes in the presynaptic region of neurons and has recently been reported to be associated with lipid rafts and caveolae. We examined the interactions between several brain sphingolipids and alpha-synuclein and found that alpha-synuclein specifically binds to ganglioside GM1-containing small unilamellar vesicles (SUVs). This results in the induction of substantial alpha-helical structure and inhibition or elimination of alpha-synuclein fibril formation, depending on the amount of GM1 present. SUVs containing total brain gangliosides, gangliosides GM2 or GM3, or asialo-GM1 had weak inhibitory effects on alpha-synuclein fibrillation and induced some alpha-helical structure, while all other sphingolipids studied showed negligible interaction with alpha-synuclein. alpha-Synuclein binding to GM1-containing SUVs was accompanied by formation of oligomers of alpha-synuclein. The familial mutant A53T alpha-synuclein interacted with GM1-containing SUVs in an analogous manner to wild type, whereas the A30P mutant showed minimal interaction. This is the first detailed report showing a direct association between GM1 and alpha-synuclein, which is attributed to specific interaction between helical alpha-synuclein and both the sialic acid and carbohydrate moieties of GM1. The recruitment of alpha-synuclein by GM1 to caveolae and lipid raft regions in membranes could explain alpha-synuclein's localization to presynaptic membranes and raises the possibility that perturbation of GM1/raft association could induce changes in alpha-synuclein that contribute to the pathogenesis of PD.