Effects of N-glycan processing inhibitors on signaling events and induction of apoptosis in galectin-1-stimulated Jurkat T lymphocytes

To elucidate the role of N-linked glycans in triggering T-cell functions, the effects of the N-glycan processing inhibitors 1-deoxymannojirimycin (1-DMM) and swainsonine (SW) were investigated on signaling events and induction of apoptosis in galectin-1 (gal-1)-stimulated Jurkat T lymphocytes. The treatment of Jurkat E6.1 cells with 1-DMM and SW strongly reduced the cell binding of gal-1-biotin, conjugate binding to cell lysate glycoproteins, and to cluster of differentiation (CD) 3 immunoprecipitates on blots as well as the binding of CD2 and CD3 to immobilized gal-1. The mannosidase inhibitors efficiently decreased gal-1-induced calcium mobilization. Both phases originated from a transient Ca(2+) release of internal stores, and the sustained influx across the plasma membrane was found to be involved. Both inhibitors suppressed in transiently transfected Jurkat T lymphocytes the gal-1-induced expression of the luciferase (luc) reporter gene constructs pNFAT-TA-Luc and pAP1(phorbol-12-myristate-13-acetate [PMA])-TA-Luc. The data provide evidence that gal-1 triggers through binding to N-linked glycans a Ca(2+)-sensitive apoptotic pathway.     

Cell calcium signalling induced by endogenous lectin carbohydrate interaction in the Jurkat T cell line

The effects of the -galactoside-binding lectin from human placenta (HPL14) on intracellular calcium concentration ([Ca²⁺]_i) were examined in the human Jurkat T cell line. The lectin induces a concentration dependent increase in [Ca²⁺]_i. This calcium signalling effect is clearly mediated through complementary cell surface galactoglycoconjugates because it can be blocked by -galactosides. The observed Ca²⁺-response involves both the release of calcium from intracellular stores and a calcium influx from the extracellular space. It is sustained in the presence of 1 mM extracellular calcium whereas it becomes transient when the influx of extracellular calcium was blocked by calcium chelation to EGTA. Voltage-sensitive calcium channel blockers like verapamil and prenylamine were without effect on the action of HPL14. Protection of the sugar binding activity of HPL14 in the absence of a thiol-reducing reagent by carboxamidomethylation (CM-HPL14) or by substitution Cys2 with serine (C2S) results in lectin proteins with considerably decreased calcium signalling efficiency. The recombinant lectin (Rec H) and the mutant protein obtained by substitution of highly conservative Trp68 with tyrosine (W68Y) induce lower levels of [Ca²⁺]_i compared to wild type lectin.Abbreviation [Ca²⁺]_i concentration of intracytoplasmic free calcium - CM carboxamidomethylation - CRD earbohydrate recognition domain - C2S mutant lectin protein in which Cys2 was replaced by serine - EGTA ethyleneglycol-bis(2-aminoethylether)-N,N,N - N-tetraacetic acid HEPES,N-(2-hydroxyethyl)piperazine-N-2-ethanesulfonic acid - HPL14 human -galactoside-binding placental lectin - Rec H recombinant human 14 kDa lectin - W68Y mutant lectin protein in which Trp68 was substituted to tyrosine     

Interaction of human MUC1 and beta-catenin is regulated by Lck and ZAP-70 in activated Jurkat T cells

The MUC1 transmembrane glycoprotein is aberrantly expressed by diverse hematologic malignancies, including those of the T cell lineage. The MUC1 cytoplasmic domain (CD) interacts with beta-catenin; however, the role of MUC1 in T cells is not known. In the present work, MUC1 was studied as a potential downstream effector of the Lck and ZAP-70 tyrosine kinases that are essential for T cell activation. The results demonstrate that anti-CD3-induced or PMA+ionomycin-induced activation of Jurkat T cells is associated with increased binding of MUC1 and Lck. Lck phosphorylates MUC1-CD on Y-46 and, in turn, stimulates the binding of MUC1 to beta-catenin. The results further demonstrate that MUC1 interacts with ZAP-70. In contrast to Lck, ZAP-70 phosphorylates MUC1-CD predominantly on Y-20. However, like Lck, ZAP-70-mediated phosphorylation of MUC1 Y-20 stimulates binding of MUC1 and beta-catenin. These findings indicate that MUC1 functions as a substrate for Lck and ZAP-70 in activated Jurkat T cells and that MUC1 integrates T cell receptor signaling with the beta-catenin pathway.     

Functional organization of TRPC-Ca channels and regulation of calcium microdomains

TRP family of proteins are components of unique cation channels that are activated in response to diverse stimuli ranging from growth factor and neurotransmitter stimulation of plasma membrane receptors to a variety of chemical and sensory signals. This review will focus on members of the TRPC sub-family (TRPC1–TRPC7) which currently appear to be the strongest candidates for the enigmatic Ca²⁺ influx channels that are activated in response to stimulation of plasma membrane receptors which result in phosphatidyl inositol-(4,5)-bisphosphate (PIP₂) hydrolysis, generation of IP₃ and DAG, and IP₃-induced Ca²⁺ release from the intracellular Ca²⁺ store via inositol trisphosphate receptor (IP₃R). Homomeric or selective heteromeric interactions between TRPC monomers generate distinct channels that contribute to store-operated as well as store-independent Ca²⁺ entry mechanisms. The former is regulated by the emptying/refilling of internal Ca²⁺ store(s) while the latter depends on PIP₂ hydrolysis (due to changes in PIP₂ per se or an increase in diacylglycerol, DAG). Although the exact physiological function of TRPC channels and how they are regulated has not yet been conclusively established, it is clear that a variety of cellular functions are controlled by Ca²⁺ entry via these channels. Thus, it is critical to understand how cells coordinate the regulation of diverse TRPC channels to elicit specific physiological functions. It is now well established that segregation of TRPC channels mediated by interactions with signaling and scaffolding proteins, determines their localization and regulation in functionally distinct cellular domains. Furthermore, both protein and lipid components of intracellular and plasma membranes contribute to the organization of these microdomains. Such organization serves as a platform for the generation of spatially and temporally dictated [Ca²⁺]_i signals which are critical for precise control of downstream cellular functions.     

Stimulation of voltage-dependent calcium channels during capacitation and by progesterone in human sperm

To fertilize, mammalian sperm must undergo two sequential steps that require activation of calcium entry mechanisms, capacitation and acrosomal exocytosis, induced in the latter case by the egg zona pellucida glycoprotein ZP3 or by progesterone. Voltage-dependent calcium channels (VDCC) could participate in these processes. Since patch clamp recordings are extremely difficult in mature sperm, the activity of VDCC has been alternatively analyzed with optical detectors of membrane potential and intracellular calcium in sperm populations. Using this approach, we previously reported that in human sperm there is a voltage-dependent calcium influx system that strongly indicates that human sperm are endowed with functional VDCC. In this study we developed evidence indicating that calcium influx through VDCC is significantly stimulated during sperm in vitro capacitation and by progesterone action, which is present in the follicular fluid that surrounds the egg. The observed effects of capacitation and progesterone on VDCC may be physiologically significant for sperm-egg interaction.     

Downregulation of TRPV6 channel activity by cholesterol depletion in Jurkat T cell line

Transient receptor potential vanilloid 6 (TRPV6) channels are key players in calcium metabolism of healthy and cancerous cells. Nevertheless, the mechanisms controlling abundance of these channels in plasma membrane of the cells to regulate Ca²⁺ transport is still poorly understood. In this study, we provide the first evidence that TRPV6 calcium channels and Ca ²⁺ influx in Jurkat T cell line are modulated by cholesterol, a main lipid component of the plasma membrane. Using patch‐clamp technique, we found that activity of TRPV6 channels decreased by cholesterol sequestration with methyl‐β‐cyclodextrin (MβCD). Continuous measurement of intracellular Ca²⁺ revealed a reduction of Ca²⁺ influx into Jurkat cells following cholesterol depletion. Immunofluorescence and immunoelectron microscopy analyses of MβCD‐treated cells detected the lower surface expression of the TRPV6 proteins in comparison with control cells. In general, our data showed that cholesterol regulates TRPV6 channel activity and TRPV6‐mediated Ca²⁺ influx in cells, apparently affecting the localization and density of the calcium channels in the plasma membrane of Jurkat T cells.     

A Novel Apoptosis-like Pathway, Independent of Mitochondria and Caspases, Induced by Curcumin in Human Lymphoblastoid T (Jurkat) Cells

We have shown previously [E. Sikora, A. Bielak-Zmijewska, K. Piwocka, J. Skierski, and E. Radziszewska (1997) Biochem. Pharmacol. 54, 899-907] that curcumin prevents formation of oligonucleosomal DNA fragmentation in rat thymocytes and human leukemic T lymphocytes (Jurkat cells) induced to undergo apoptosis. In this paper we show that 50 microM curcumin by itself induces cell death in Jurkat cells, but its symptoms differ from those observed after a short ultraviolet (uv) irradiation. Ultraviolet-irradiated Jurkat cells displayed typical symptoms of apoptosis: morphological changes, internucleosomal and high-molecular-weight DNA fragmentation, formation of sub-G1 fractions in DNA content frequency histograms, and dissipation of the mitochondrial transmembrane electric potential (Delta psi). In contrast, curcumin-treated Jurkat cells exhibited DNA splitting into high-, but not low-, molecular-weight fragments. These cells retained their high mitochondrial Delta psi, and the content of Ca2+ in endoplasmic reticulum stores remained at the level typical for untreated cells. The frequency of opening of the mitochondrial permeability transition pores in curcumin-treated cells was decreased compared to the controls, whereas uv irradiation made these pores completely open. Curcumin did not produce any change in the activity of caspase-3, whereas uv irradiation considerably activated this protease. The morphology of curcumin-treated cells displayed chromatin condensation, which was insensitive to the caspase inhibitor z-VAD-fmk, but no formation of typical apoptotic bodies, as was the case after uv irradiation. In contrast to uv-irradiated cells, curcumin-treated Jurkat cells considerably increased the level of Bcl-2. It is concluded that the programmed cell death induced by curcumin in Jurkat cells differs from "classical" by the lack of mitochondrial depolarization and of the involvement of caspases.     

Galectin-1 induced activation of the apoptotic death-receptor pathway in human Jurkat T lymphocytes

Galectin-1 (gal-1), a member of the family of β-galactoside binding proteins, participates in several biological processes such as immunomodulation, cell adhesion, regulation of cell growth and apoptosis. The aim of this study was to investigate whether gal-1 interferes with the Fas (Apo-1/CD95)-associated apoptosis cascade in the T-cell lines Jurkat and MOLT-4. Gal-1 and an Apo-1 monoclonal antibody (mAb) induced DNA-fragmentation in Jurkat T-cells whereas MOLT-4 cells were resistant. Gal-1 stimulated DNA-fragmentation could be efficiently inhibited by caspase-8 inhibitor II (Z-IETD-FMK) and a neutralizing Fas mAb. Fas could be identified as a target for gal-1 recognition as demonstrated by immunofluorescence staining, binding of the receptor glycoprotein to immobilized gal-1 and analyses by immunoblotting as well as by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gal-1 stimulates the activation and proteolytic processing of procaspase-8 and downstream procaspase-3 in Jurkat-T cells. Inhibition of gal-1 induced procaspase-8 activation by a neutralizing Fas mAb strongly suggests that gal-1 recognition of Fas is associated with caspase-8 activation. Our data provide the first experimental evidence for targeting of gal-1 to glycotopes on Fas and the subsequent activation of the apoptotic death-receptor pathway.     

Ceramide increase cytoplasmic Ca2+ concentration in Jurkat T cells by liberation of calcium from intracellular stores and activation of a store-operated calcium channel

The effect of ceramide on the cytoplasmic Ca2+ concentration ([Ca2+]i) varies depending on the cell type. We have found that in Jurkat human T cells ceramide increases the [Ca2+]i from a thapsigargin-sensitive calcium pool and the subsequent activation of a capacitative Ca2+ entry. This effect occurs both in the presence and in the absence of extracellular calcium. Addition of ceramine, a non-hydrolysable analogue of ceramide, reproduced its effect on the [Ca2+]i ruling out that this is due to the conversion of ceramide to sphingosine. The effect of ceramide was additive to that obtained by sphingosine, but not to the Jurkat T cells specific antibody OKT3. However, different to the latter, ceramide do not induced an elevation of InsP3. The opening of a store operated Ca2+ channel by ceramide was corroborated by experiments of Fura-2 quenching, using Mn2+ as a surrogate for Ca2+ and confirmed by whole-cell recording patch clamp techniques.     

超抗原刺激Jurkat T细胞的原子力显微镜研究

利用原子力显微镜分别对未加药人急性T淋巴细胞性白血病细胞（Jurkat细胞）和经超抗原刺激不同时间（6,12,48,72 h）后的Jurkat 细胞进行了细胞全貌和细胞膜表面纳米结构成像和探测研究,并通过比较不同状态下细胞表面的粘附力变化,探讨Jurkat细胞形态变化与粘附行为之间的关系,用CCK-8检测细胞的增殖,以期对Jurkat 细胞形态结构和细胞功能之间的关系有进一步的了解。结果发现：与未加药的Jurkat 细胞相比,随着超抗原刺激时间的延长,Jurkat 细胞的体积、高度、半宽度、粗糙度等参数发生明显的变化;活化48、72 h时,细胞与针尖间的相互作用力大约是活化6 h时的5倍,活化过程中细胞膜表面纳米结构的改变,引起其机械性能的变化。Jurkat 细胞的表面超微结构、细胞膜结构的改变和分化对于更深入地了解T细胞活化与免疫信号的传递,阐明其免疫过程的作用机制具有重要的意义。     

Involvement of protein kinase C-alpha and -epsilon in extracellular Ca signalling mediated by the calcium sensing receptor

The sensing of extracellular Ca²⁺ concentration ([Ca²⁺]_o) and modulation of cellular processes associated with acute or sustained changes in [Ca²⁺]_o are cell-type specific and mediated by the calcium sensing receptor (CaR). [Ca²⁺]_o signalling requires protein kinase C (PKC), but the identity and role of PKC isoforms in CaR-mediated responses remain unclear. Here we show that high [Ca²⁺]_o activated PKC-α and PKC-ε in parathyroid cells and in human embryonic kidney (HEK293) cells overexpressing the CaR (HEK-CaR) and that this response correlated with the CaR-dependent activation of mitogen-activated protein kinases ERK1/2. Activation of ERK1/2 by acute high [Ca²⁺]_o required influx of Ca²⁺through Ni²⁺-sensitive Ca²⁺channels and phosphatidylinositol-dependent phospholipase C-β activity. Inhibition of PKC by co-expression of dominant-negative (DN) mutants of PKC-α or -ε with the CaR attenuated sustained ERK1/2 activation. Overexpression of a PKC phosphorylation site (T888A) mutant CaR in HEK293 cells showed that this site was important for ERK1/2 activation at high [Ca²⁺]_o. Activation of ERK1/2 by high [Ca²⁺]_o was not necessary for the [Ca²⁺]_o-regulated secretion of parathyroid hormone (PTH) in dispersed bovine parathyroid cells. These data suggest that the CaR-mediated [Ca²⁺]_o signal leading to regulated PTH secretion that requires diacylglycerol-responsive PKC isoforms is not mediated via the ERK pathway.     

Store-operated calcium entry and non-capacitative calcium entry have distinct roles in thrombin-induced calcium signalling in human platelets

Phosphatidylserine (PS)-exposing platelets accelerate coagulation at sites of vascular injury. PS exposure requires sustained Ca²⁺ signalling. Two distinct Ca²⁺ entry pathways amplify and sustain platelet Ca²⁺ signalling, but their relative importance in human platelets is not known. Here we examined the relative roles of store-operated Ca²⁺ entry (SOCE) and non-capacitative Ca²⁺ entry (NCCE) in thrombin-induced Ca²⁺ signalling and PS exposure by using two Ca²⁺ channel blockers. BTP-2 showed marked selectivity for SOCE over NCCE. LOE-908 specifically blocked NCCE under our conditions. Using these agents we found that SOCE is important at low thrombin concentrations whereas NCCE became increasingly important as thrombin concentration was increased. PS exposure was reduced by LOE-908, and only activated at thrombin concentrations that also activate NCCE. In contrast, BTP-2 had no effect on PS exposure. We suggest that SOCE amplifies and sustains Ca²⁺ signalling in response to low concentrations of thrombin whereas both NCCE and SOCE are important contributors to Ca²⁺ signalling at higher thrombin concentrations. However, despite being involved in Ca²⁺ signalling at high thrombin concentrations, SOCE is not important for thrombin-induced PS exposure in human platelets. This suggests that the route of Ca²⁺ entry is an important regulator of thrombin-induced PS exposure in platelets.     

Antiapoptotic role of p38 mitogen activated protein kinase in Jurkat T cells and normal human T lymphocytes treated with 8-methoxypsoralen and ultraviolet-A radiation

A combination of 8-methoxypsoralen and ultraviolet-A radiation (320–400 nm) (PUVA) is used for the treatment of T cell-mediated disorders, including chronic graft-versus-host disease, autoimmune disorders, and cutaneous T-cell lymphomas. The mechanisms of action of this therapy, referred to as extracorporeal phototherapy, have not been fully elucidated. PUVA is known to induce apoptosis in T lymphocytes collected by apheresis, however no information is available concerning the underlying signaling pathways which are activated by PUVA. In this study, we found that PUVA treatment of Jurkat cells and human T lymphocytes up-regulates the p38 MAPK pathway but not the p42/44 MAPK or the SAPK/JNK signaling networks. The use of a pharmacological inhibitor selective for the p38 MAPK pathway, SB203580, allowed us to demonstrate that this network exerts an antiapoptotic effect in PUVA-treated Jurkat cells and T lymphocytes from healthy donors. Moreover, the effect of SB203580 was not due to a down-regulation of the Akt survival pathway which was not activated in response to PUVA. These results may suggest that p38 MAPK-dependent signaling is very important for the regulation of survival genes after exposure to PUVA. Since the therapeutic effect of PUVA seems to depend, at least in part, on apoptosis, further studies on the apoptosis signaling networks activated by this treatment might lead to the use of signal transduction modulators in combination with PUVA, to increase the efficacy of this form of therapy.     

Orai1-NFAT signalling pathway triggered by T cell receptor stimulation

T cell receptor (TCR) stimulation plays a crucial role in development, homeostasis, proliferation, cell death, cytokine production, and differentiation of T cells. Thus, in depth understanding of TCR signalling is crucial for development of therapy targeting inflammatory diseases, improvement of vaccination efficiency, and cancer therapy utilizing T cell-based strategies. TCR activation turns on various signalling pathways, one of the important one being the Ca²⁺-calcineurin-nuclear factor of activated T cells (NFAT) signalling pathway. Stimulation of TCRs triggers depletion of intracellular Ca²⁺ store and in turn, initiates store-operated Ca²⁺ entry (SOCE), one of the major mechanisms to raise the intracellular Ca²⁺ concentrations in T cells. Ca²⁺-release-activated-Ca²⁺ (CRAC) channels are a prototype of store-operated Ca²⁺ (SOC) channels in immune cells that are very well characterized. Recent identification of STIM1 as the endoplasmic reticulum (ER) Ca²⁺ sensor and Orai1 as the pore subunit has dramatically advanced the understanding of CRAC channels and provides a molecular tool to investigate the physiological outcomes of Ca²⁺ signalling during immune responses. In this review, we focus on our current understanding of CRAC channel activation, regulation, and downstream calcineurin-NFAT signaling pathway.     

Caspase-1 Is Not Involved in CD95/Fas-Induced Apoptosis in Jurkat T Cells

It is now well established that the caspases, a family of cysteine proteases, play a key role in apoptosis. Although overexpressing each of the caspases in cells triggered apoptosis, the precise role and contribution of individual caspases are still unclear. Caspase-1, the first caspase discovered, was initially implicated in mammalian apoptosis because of its similarity to the gene productced-3.Using whole cells as well as anin vitrosystem to study apoptosis, the role of caspase-1 in Fas-mediated apoptosis in Jurkat T cells was examined in greater detail. Using various peptide-based caspase inhibitors, our results showed thatN-acetyl-Tyr-Val-Ala-Asp chloromethyl ketone and benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone efficiently blocked Fas-mediated apoptosis in Jurkat T cells, whereasN-acetyl-Tyr-Val-Ala-Asp aldehyde, which is more specific for caspase-1, had little effect. Cell lysates derived from anti-Fas-stimulated cells, which readily induced apoptotic nuclei morphology and DNA fragmentation in isolated thymocyte nuclei, had no caspase-1 activity using proIL-1β as a substrate. Time-course studies showed no caspase-1 activity during the activation of apoptosis in Jurkat cells by agonistic Fas antibodies. Furthermore, no pro-caspase-1 protein nor activated form of the protein was detected in normal or apoptotic Jurkat cells. In contrast, both caspase-2 and caspase-3 were readily detected as proenzymes in control cells and their activated forms were detected in apoptotic cells. Incubation of recombinant active caspase-1 with control cell lysates did not activate the apoptotic cascade as shown by the lack of detectable apoptotic nuclei promoting activity using isolated nuclei as substrate. However, under similar conditions proIL-1β was readily processed into the mature cytokine, indicating that the recombinant caspase-1 remained active in the presence of control cell lysates. Taken together our results demonstrate that caspase-1 is not required for the induction of apoptosis in Jurkat T cells mediated by the Fas antigen.     

Mechanisms of transmembrane signalling in human T cell activation

Several monoclonal antibodies directed against a number of T cell surface molecules are used to elucidate the role of these molecules (cell surface molecules) in T cell activation. The activation of T cells via these molecules are both antigen-dependent (CD3/TcR complex) and antigen-independent. Irrespective of their antigen-dependency, these monoclonal antibodies activate T cells by a classical signal transduction pathway, in which the binding of monoclonal antibodies to their cell surface receptors leads to activation of phospholipase C resulting in the the depolarization of plasma membrane, hydrolysis of IP2 and IP3 and DAG, the second messengers. IP3 leads to mobilization of intracellular calcium to contribute to an increase in [Ca⁺⁺]_i, whereas DAG causes activation and translocation of PKC and an increasing apparent affinity for Ca⁺⁺. The role of IN in the mobilization of intracellular calcium is emerging. In addition, influx of extracellular calcium also contributes to increase in [Ca^–+];. The increase in [Ca⁺⁺]; following activation via some T cell surface antigen is predominantly due to intracellular mobilization of Ca^–+ (e.g. CD3/TcR complex), whereas activation via other T cell surface antigen, the increase in [Ca^+–]_i is almost entirely due to an influx of extracellular calcium (e.g. CD5 antigen). All these molecules activate autocrine system of T cell growth, namely IL-2 production, IL-2 receptor expression and T cell proliferation.     

Regulation of the type III InsP(3) receptor by InsP(3) and calcium

It has been proposed that the inositol 1,4,5-trisphosphate receptor (InsP(3)R) type III acts as a trigger for InsP(3)-mediated calcium (Ca(2+)) signaling, because this InsP(3) isoform lacks feedback inhibition by cytosolic Ca(2+). We tested this hypothesis in RIN-m5F cells, which express predominantly the type III receptor. Extracellular ATP increases Ca(2+) in these cells, and we found that this effect is independent of extracellular Ca(2+) but is blocked by the InsP(3)R antagonist heparin. There was a dose-dependent increase in the number of cells responding to ATP and two-photon flash photolysis of caged-Ca(2+) heightened the sensitivity of RIN-m5F cells to this increase. These findings provide evidence that Ca(2+) increases the sensitivity of the InsP(3)R type III in intact cells and supports the idea that this isoform can act as a trigger for hormone-induced Ca(2+) signaling.