From the Department of Immunology and Cellular
Biology, Research Center Borstel, D-23845 Borstel, Germany, the
Department of Food Science, University of Guelph, Guelph,
Ontario N1G 2W1, Canada, the ** Department of Microbiology
and Immunology, Stritch School of Medicine, Loyola University Chicago,
Maywood, Illinois 60153, and the
Department of Dermatology, University
Hospital Eppendorf, University of Hamburg,
D-20246 Hamburg, Germany
Received for publication, June 26, 2002, and in revised form, September 26, 2002
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ABSTRACT |
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ATP-gated ion channel P2X receptors
are expressed on the surface of most immune cells and can trigger
multiple cellular responses, such as membrane permeabilization,
cytokine production, and cell proliferation or apoptosis. Despite broad
distribution and pleiotropic activities, signaling pathways downstream
of these ionotropic receptors are still poorly understood. Here, we
describe intracellular signaling events in Jurkat cells treated with
millimolar concentrations of extracellular ATP. Within minutes, ATP
treatment resulted in the phosphorylation and activation of
p56lck kinase, extracellular signal-regulated kinase (ERK), and
c-Jun N-terminal kinase but not p38 kinase. These effects were
wholly dependent upon the presence of extracellular
Ca2+ ions in the culture medium. Nevertheless,
calmodulin antagonist calmidazolium and CaM kinase inhibitor KN-93 both
had no effect on the activation of p56lck and ERK, whereas a
pretreatment of Jurkat cells with MAP kinase kinase inhibitor P098059
was able to abrogate phosphorylation of ERK. Further, expression of
c-Jun and c-Fos proteins and activator protein (AP-1) DNA binding
activity were enhanced in a time-dependent manner. In
contrast, DNA binding activity of NF- Extracellular ATP and other nucleotides act through specific cell
surface receptors and can regulate a variety of cellular responses in
many cell types and tissues (1-7). Among them are such different
phenomena as platelet aggregation, smooth muscle contractility,
excitatory transmitter function, mitogenic stimulation, or induction of
cell death (reviewed in Refs. 1 and 8). The biological effects of
extracellular nucleotides are mediated via stimulation of two primary
classes of purinergic receptors, P1 and P2. The P1 receptors are
responsive to adenosine, whereas the P2 receptors respond to a variety
of nucleotides, including ATP (7, 9). The P2 receptors are subdivided
in two mechanistically distinct subclasses, the metabotropic G
protein-coupled P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11-13)
and the ionotropic ligand-gated channel P2X receptors (P2X1-7)
(9-12). Activation of the P2Y receptors generally induces downstream
signaling through the G protein-coupled activation of phospholipase C,
followed by Ca2+ mobilization from intracellular stores
(13, 14). The P2Y11 also activates adenylyl cyclase, whereas the P2Y12
inhibits it (15). The seven subunits of the ionotrophic ATP-gated P2X
receptor family comprise a different subclass, ranging from 379 to 595 amino acids in length and regulating the intracellular level of Ca2+ by ligand-stimulated increase in cell membrane
permeability for extracellular Ca2+ ions (7, 16, 17).
The P2X7 receptor is a 595-amino acid polypeptide and has a
structure similar to that of other P2X receptors with two
membrane-spanning domains, a large extracellular loop, and
intracellular N- and C-terminal domains (18, 19). In contrast to other
P2X receptors, the P2X7 C-terminal intracellular chain is about 200 amino acids longer (20, 21). The P2X7 has a pharmacological profile
similar to the receptor previously designated as P2Z, with prominent
expression in many immune cells (lymphocytes, monocytes/macrophages,
dendritic, mesangial, and microglial cells) (7, 20), and requires
millimolar levels of ATP in the presence of divalent cations to achieve
activation (22, 23). Although each of the P2X receptors is capable of forming heteromers with other family members in a specific pattern (24), the P2X7 cannot heteropolymerize with any other P2X subunit (25).
Activation of the P2X7 receptor results in the formation of a
nonselective cationic channel with low affinity for ATP and increased
permeability to Ca2+, intracellular depolarization, and
equilibration of sodium and potassium gradients (18, 26). In addition,
the P2X7 receptor may also induce a nonselective pore with
uncharacterized structure able to pass molecules up to 900 Da, sharing
this ability, albeit to a lesser degree, with other P2X family members
(7, 27). Continuous activation of the receptor and the formation of a
large transmembrane pore can cause perturbations in ion homeostasis and
finally result in cell death (20, 28). Depending on the cell
background, stimulation of the P2X7 receptor by ATP triggers diverse
biological responses, such as post-translational processing of
precursors of IL-1 The activation of protein-tyrosine kinases constitutes one of the
initial steps for the induction of signaling cascades, which ultimately
results in the activation of T cell effector function (35).
p56lck kinase is a lymphoid-specific cytoplasmic
protein-tyrosine kinase with a molecular size of about 56 kDa that
mediates initial events in TCR/CD3 signaling, such as
phosphorylation of the TCR complex within amino acid sequences known as
immunoreceptor-based tyrosine activation motifs (35, 36), which serve
as docking sites for Src homology domain 2-containing molecules,
predominantly Zap-70 and Syk (35, 37), and activation of
mitogen-activated protein (MAP) kinases (38). The MAP kinase cascade
represents another key signaling pathway, critical for the linking
membrane receptors to cytoplasmic and nuclear effectors. Extracellular
signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38
are serine/threonine kinases, which constitute major components of this
inducible signaling pathway and regulate many intracellular events,
including cell proliferation and differentiation (39, 40). Upon
activation, the MAP kinases phosphorylate various cytoplasmic effector
proteins and are translocated to the nucleus, where they participate in the regulation of the gene expression by acting on transcription factors (39, 41).
The present study focuses on intracellular signaling events in Jurkat
cells treated with millimolar concentrations of extracellular ATP. We
show here that ATP induces phosphorylation and activation of
p56lck kinase, ERK, and JNK. These events are prerequisite for
the subsequent increase in the expression of c-Jun and c-Fos proteins
and activation of transcription factor AP-1. On the contrary, DNA
binding activity of p50 and p65 (RelA) subunits of NF- Reagents and Antibodies--
All of the reagents used were of
analytical grade. ATP was obtained from Roche Molecular Biochemicals.
PD098059, an inhibitor of MEK (42); suramine, an antagonist of the P2
receptors (43); calmidazolium, an antagonist of calmodulin (44); KN-93,
an inhibitor of Ca2+/calmodulin-dependent
protein kinase II (CaM kinase) (44); and KN-92, an inactive analog of
KN-93, were purchased from Calbiochem-Novabiochem (La Jolla, CA).
1[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62) and oxidized ATP (oATP), both antagonists of the P2X7 receptor
(7, 45, 46); 3-O-(4'-benzoyl)-benzoyl-benzoyl-ATP (Bz-ATP),
an agonist of the P2X7 receptor (7); and UTP, GTP, adenosine, and
calcium ionophore A23187 were purchased from Sigma. Anti-Tyr(P)
antibodies (horseradish peroxidase-conjugated (RC20H) and biotinylated
(RC20B)) were obtained from Transduction Laboratories (Lexington, KY).
Antibodies against ERK (C-16), pERK (E-4), pJNK (G-7), pp38 (D-8), Lck
(3A5), NF- Cell Culture and Stimulation Conditions--
Human
T-lymphoblastoid cell line Jurkat, its p56lck-deficient
derivative variant JCaM1, and stable transfectants of JCaM1 expressing p56lck cDNA in pBP1 vector or mock pBP1 vector (last three
lines were kindly provided by Dr. D. Straus, University of Chicago)
(36) were maintained in RPMI 1640, supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 mg/ml streptomycin.
Transfectants were maintained in the presence of 0.5 mg/ml of G418 and
0.5 mg/ml hygromycin. Before treatment the cells were washed twice with Dulbecco's phosphate-buffered saline and incubated in RPMI 1640 without fetal calf serum at 37 °C for 3 h.
The cells (5 × 106) were activated by ATP (final
concentration, 3 mM) for different time intervals at
37 °C. Activation was interrupted by adding ice-cold
phosphate-buffered saline with 10 mM EDTA and 100 mM sodium vanadate. Then the cells were pelleted and stored
at Immunoprecipitation, Western Blotting, and Kinase Assay--
The
cell pellets were lysed in 1% Nonidet P-40 cell extraction buffer (20 mM Tris-HCl buffer, pH 8.0, with 15 mM NaCl,
1% Nonidet P-40, 2 mM EDTA, 1 mg/ml pepstatin A, 1 mg/ml
leupeptin, 10 mM phenylmethylsulfonyl fluoride, and 100 mM sodium vanadate). The detergent-insoluble material was
removed by centrifugation for 15 min at 13.000 and 4 °C. Protein
concentrations were determined using a bovine serum albumin protein
assay kit (Bio-Rad), and 50 µg of proteins were analyzed by
electrophoresis in 10% SDS-PAGE. For immunoprecipitation, 500 µg of
proteins were incubated overnight at 4 °C with 5 µg/ml of RC20B or
anti-Lck antibodies. Immunocomplexes were captured on protein A-agarose
(Bio-Rad) or streptavidin-agarose (Pierce), respectively, with gentle
mixing for 1 h at 4 °C and analyzed in 10% SDS-PAGE.
Visualization of specific proteins was carried out by an ECL method
using ECL Western blotting detection reagents (Amersham Biosciences)
according to the manufacturer's recommendations.
For kinase assay, immunocomplexes were washed with 25 mM
HEPES (pH 7.4), 2 mM MnCl2, 10 mM
MgCl2, 1 mM Na3VO4, and
incubated in 60 µl of 5 mM HEPES, 2 mM
MnCl2, 10 mM MgCl2, 1 mM Na3VO4, 10 µCi of
[ RNA Extraction and RT-PCR--
Cellular RNA was extracted from
the cells using TRIZOL reagent (Invitrogen) according to the
manufacturer's instructions. A 5-µg aliquot of total cellular RNA
was reverse transcribed using random hexanucleotides as primers and a
Superscript II preamplification kit (Invitrogen). cDNA was
amplified in 50 µl of PCR reaction mixture, containing 250 µM of each dNTP, 200 nM primers, 5 µl of
10-fold PCR buffer with 1.5 mM MgCl2 and 1 unit
of Taq DNA polymerase (Amplitaq; Applied Biosciences,
Warrington, UK). The primers used were: human P2X7 receptor
(GenBankTM accession number Y09561): sense,
5'-TCCGAGAAACAGGCGATAA-3', and antisense, 5'-ACTCGCACTTCTTCCTGTA-3';
human IL-2 (GenBankTM accession number NM 000586):
sense, 5'-TACAACTGGAGCATTTACTGC-3', and antisense,
5'-TTGACAAAAGGTAATCCATCT-3'; human Electromobility Shift Assay (EMSA)--
Nuclear extracts from
2 × 106 cells were prepared according to the method
of Schreiber et al. (47). EMSA was performed by incubating 4 mg of nuclear extract with 16 fmol of 32P end-labeled
double-stranded NF- Proliferation Assay, Cell Survival Assay, and Flow Cytometric
Analysis--
Proliferation of Jurkat cells was assessed by
[3H]thymidine incorporation. 1 × 105
cells/well were cultured in triplicates in 96-well flat bottom plates
in a final volume of 200 µl for 24 h and then incubated with
[3H]thymidine (1 µCi/well) for an additional 12 h.
The cells were harvested onto glass filters, and incorporation of
[3H]thymidine was determined by liquid scintillation counting.
For cell survival assays, Jurkat cells were cultured at 1 × 105 cells/ml in triplicate at 37 °C for 1-3 days in
RPMI medium in the presence of 3 mM ATP. The cells were
harvested, and cell viability was determined by fluorescence-activated
cell sorter analysis with 5 µg/ml of propidium iodide. The percentage
of apoptotic cells was evaluated by an ApoTarget annexin V-fluorescein
isothiocyanate apoptosis kit (BioSourse Int.) according to the
manufacturer's protocol and analyzed on a FACScalibur (Becton
Dickinson, San Jose, CA) using CELLQuest software.
Intracellular Ca2+ Measurement--
The cells were
preincubated with 2 µM of Fura-2 (Molecular Probes,
Eugene, OR) for 30 min at 37 °C, and Ca2+ influx was
measured in Hitachi F-2500 spectrophotometer (Hitachi, Tokyo, Japan)
using 350/385 excitation filters. After recording of background for
20 s, the cells were stimulated with 3 mM ATP, and the
concentration of Ca2+ was calculated using the equation of
Grinkievich et al. (48).
Changes in Plasma Membrane
Permeability--
ATP-dependent increases in plasma
membrane permeability were measured with the extracellular fluorescent
tracers Lucifer yellow (Molecular Probes) and ethidium bromide (Sigma),
as described earlier (25). For Lucifer yellow uptake, the cells were
incubated for 15 min at 37 °C in buffer (125 mM NaCl, 5 mM KCl, 1 mM MgSO4, 1 mM Na2HPO4, 5.5 mM
glucose, 5 mM NaHCO3, 20 mM HEPES,
pH 7.4) containing 250 µM sulfinpyrazone and 1 mg/ml
Lucifer yellow and stimulated with 3 mM of ATP. After
several washings to remove the extracellular dye, the cells were
analyzed with an inverted fluorescence microscope (Nikon Diaphot 300, Tokyo, Japan) using a 40× objective and a fluorescein filter. For
ethidium bromide uptake, the cells were incubated in a fluorometer
cuvette (37 °C) at a concentration of 106 cells/ml in
the presence of 20 µM ethidium bromide and challenged with various ATP concentrations. The fluorescence changes were monitored at the wavelength 360-580 nm.
Statistical Analysis--
All of the experiments were performed
in at least three independent assays, which yielded highly comparable
results. The data are presented as the mean values ± S.D. as
indicated in the figure legends. The Mann-Whitney-U test was
used to determine the level of statistical significance.
Extracellular ATP Induces Activation of p56lck
Kinase--
Protein-tyrosine kinases are critically involved in
signaling pathways that regulate cell growth, differentiation, and
activation. The activation of downstream signaling events, including
the Ras/Raf/MAP kinase pathway, the phosphatidylinositol pathway, and
the phosphatidylinositol 3-kinase pathway, was shown to be dependent
upon protein-tyrosine kinase functions (39, 49). Thus, we studied first
the ability of purinoreceptors to mediate intracellular signaling in
response to extracellular ATP by incubating Jurkat cells with various
concentrations of ATP for different time intervals and evaluating the
phosphorylation of protein-tyrosine kinases. In the experiments
illustrated in Fig. 1, Jurkat cells were
treated with 3 mM ATP. Immunoprecipitation and probing with
anti-Tyr(P) antibodies at the indicated time points showed that a
number of proteins were phosphorylated upon the exposure of the cells
to extracellular ATP. These included 40-42-, 56-, 70-, and 80-85-kDa
proteins (Fig. 1A). Such a phosphorylation pattern was
already detectable within the first 5 min of ATP administration, reaching its maximum at 15 min and returning to a near basal level within 30 min. Reportedly, T cell activation often involves the recruitment of p56lck kinase, which is essential for T cell
development and function (36). Reprobing of the blots with
anti-p56lck-specific antibodies allowed us to identify among
the phosphorylated proteins p56lck kinase (Fig. 1B).
Other kinases of the Src family, such as c-Src, Lyn, and Fyn as well as
Syk and ZAP-70, which are often recruited for signal transduction in T
cells (50), were not involved (data not shown). To confirm the
phosphorylation of p56lck kinase upon ATP administration,
immunoblotting of anti-p56lck precipitates with anti-Tyr(P)
antibodies was carried out (Fig. 1C). As shown in the
picture, ATP induced transient phosphorylation of p56lck within
5-15 min of treatment, and this effect significantly declined within
15-30 min. For loading control, the stripped membranes were reprobed
with anti-p56lck antibodies (Fig. 1D). Thus, the
kinetic experiments revealed that ATP stimulated the rapid and
transient phosphorylation of p56lck kinase.
According to several reports, the activity of p56lck is
regulated by phosphorylation at two tyrosine residues,
Tyr394 and Tyr505 (51-53).
Phosphorylation at Tyr505 leads to stabilization of
p56lck in a biologically inactive conformation (51, 52). On the contrary, phosphorylation at Tyr394 stimulates the
catalytic activity of p56lck (52, 53). We immunoprecipitated
p56lck and probed the membrane with phosphospecific antibodies
to Tyr416 of human c-Src (which corresponds to
Tyr394 in p56lck) (53) and with antibodies to
Tyr505-Lck to detect which tyrosine residues are
phosphorylated in p56lck kinase after exposure to extracellular
ATP. As shown in Fig. 2A, ATP
induced the phosphorylation of Tyr394 (top
panel) but did not change the phosphorylation of
Tyr505 (middle panel).
Because phosphorylation of Tyr394 usually leads to an
increase in the kinase activity of p56lck (53), a kinase assay
was performed to determine whether ATP also affects p56lck
kinase catalytic activity. We incubated p56lck-precipitates
with [ Extracellular ATP Activates the MAP Kinases ERK1, ERK2, and
JNK1/2 but not p38--
It was shown that ATP can induce
phosphorylation of ERK in PC12 cells and fetal astrocytes (54-56) and
of JNK in BAC1 murine macrophages (26). Several studies reported an
important role for p56lck kinase in the activation of the MAP
kinase pathway (38, 57). Based on these findings, our next goal was to
determine whether ATP is able to induce the activation of ERK and JNK
MAP kinases in Jurkat cells. The cells were incubated with ATP, and the
activation of the MAP kinases ERK and JNK was evaluated by employment
of phosphospecific antibodies directed against the C termini of ERK1 and ERK2 (both phosphorylated at Tyr204) (55) and JNK1 and
JNK2 (phosphorylated at Thr183 and Tyr185,
respectively) (40). Kinetic experiments revealed that ATP induced rapid
and transient phosphorylation and activation of ERK1 (p44) and ERK2
(p42), as well as JNK1 (p54) and JNK2 (p52) in a
time-dependent manner (Fig.
3). Phosphorylation of both kinases was
already detectable within 5 min of ATP administration, reaching its
maximum after 15 min and returning to near basal levels within 3 h. On the other hand, the phosphorylation state of p38 kinase was not
affected, remaining at the same level throughout the time course of the
experiment (Fig. 3). For loading control, the membranes were stripped
and reprobed with anti-ERK, anti-JNK, or anti-p38 antibodies,
respectively. The activation of MAP kinases by ATP required a sustained
increase in the intracellular Ca2+ concentration and
Ca2+ influx across the plasma membrane, because it was
inhibited by the depletion of extracellular Ca2+ ions in
extracellular medium (data not shown).
To investigate the mechanism of activation of ERK, we examined the
effects of a specific inhibitor of MAP kinase kinase (MEK), PD098059
(42). The ERK activation cascade is believed to proceed through
sequential activation of three protein kinases, MAP kinase kinase
kinase, MEK, and MAP kinase (ERK). The MEK1/2 activates ERK by
phosphorylation of the conserved Thr and Tyr residues. PD098059
inhibits activation of both MEK1 (IC50 = 5-10
µM) and MEK2 (IC50 = 50 µM)
(42). Thus, we used PD098059 to study whether it can block
phosphorylation of ERK in response to the ATP treatment. The cells were
incubated with 50 µM of PD098058 for 90 min and then
stimulated with ATP. Untreated cells were used as a control. As shown
in Fig. 4A, PD098059 was able
to inhibit the phosphorylation of ERK (top panel). The
second panel shows the control of loading. At the same time,
the phosphorylation pattern of p56lck was not altered. The
lysates from PD098059-treated and ATP-activated cells were precipitated
with anti-p56lck antibodies, and phosphorylation of
p56lck at Tyr394 was evaluated by SDS-PAGE using
anti-pY416(c-Src) specific antibodies. As shown in Fig. 4A
(third panel), PD098059 did not affect ATP-mediated phosphorylation of p56lck kinase. For control of loading, these
membranes after stripping were probed with anti-p56lck
antibodies (Fig. 4A, bottom panel).
Recently, Franklin et al. (44) demonstrated that calcium
ionophore-induced Ca2+ influx is able to trigger a rapid
activation of ERK in the similar cell model through CaM
kinase-dependent stimulation of p56lck. To test the
involvement of calmodulin and CaM kinase in ATP-mediated activation of
p56lck and ERK1/2, Jurkat cells were pretreated with an
antagonist of calmodulin, calmidazolium, or an inhibitor of CaM kinase,
KN-93 (44), for 30 min at 37 °C, stimulated with 3 mM
ATP for different time intervals (5-15 min), and analyzed for the
activation of ERK1/2 and p56lck. Calcium ionophore A23187 and
an inactive analog of KN-93, KN-92, were used as controls. As shown in
Fig. 4B (left panel), ATP alone induced a rapid
activation of ERK1/2 after 5 min of treatment, with a peak within 15 min. In agreement with the previous report (44), A23187 also was able
to trigger activation of ERK. However, calmidazolium at a concentration
of 5 µM was able to abrogate A23187-induced but not
ATP-induced activation of ERK1/2 (Fig 4B, upper left
panel). The second panel shows control of
loading. Then we immunoprecipitated p56lck and probed the
membrane with phosphospecific antibodies to detect the activation of
p56lck. As shown in Fig. 4B, ATP induced the
phosphorylation and activation of the kinase in a
time-dependent manner both in the absence and presence of
calmidazolium, clearly indicating that ATP-mediated activation of
p56lck is not dependent on calmodulin. The lower membrane was
probed with anti-p56lck antibodies to prove the control of
loading. The experiments with CaM kinase inhibitor KN-93 revealed that
although ATP preserved the ability to activate both p56lck and
ERK1/2 (Fig. 4B, right panels), this effect was
slightly diminished when compared with its inactive analog, KN-92.
Lower membranes show the control of loading. Notably, KN-93 was able to
completely inhibit the activation of ERK and p56lck in response
to calcium ionophore A23187, whereas KN-92 had no effect. The
difference between action of KN-93 and KN-92 on Jurkat cells after
stimulation with ATP deserves additional exploration and may reflect
the partial involvement of CaM kinase to the observed activation of
p56lck and ERK1/2 in response to ATP, although this speculation
requires further proof. Nevertheless, the ability of ATP to induce the activation of ERK1/2 and p56lck kinase even in the presence of
CaM kinase inhibitor KN-93 strongly supports the idea that such an
effect is elicited mainly via a CaM kinase-independent mechanism.
Taken together, these results confirm the ability of extracellular ATP
to trigger the phosphorylation and activation of ERK1/2 and JNK1/2 but
not p38 kinase and show the contribution of MEK to the activation of
ERK. In addition, such activation of p56lck kinase and ERK1/2
appears not to be dependent on calmodulin and only partially, if at
all, dependent on CaM kinase.
Activation of p56lck and MAP Kinases by ATP Is Mediated
through the P2X7 Receptors--
To identify both the specificity of
the response and the identity of the receptors involved in ATP-induced
activation of p56lck and MAP kinases in Jurkat cells, a panel
of purinoreceptors was tested for their ability to activate these
kinases. Each purinoreceptor subtype is defined by its relative
response to different purinergic ligands (7, 9). In our preliminary
experiments, we did not observe the activation of p56lck and
MAP kinases in response to the treatment with adenosine, ADP, AMP, GTP,
or UTP (data not shown). Adenosine has been demonstrated to be a
selective agonist of P1 receptors (13). UTP serves as a high potency
agonist for human P2Y2 and P2Y4 receptors, whereas at P2Y1 and P2Y11 it
is inactive (7). ADP activates P2Y12 and P2Y13 and was reported to be
equipotent or even more potent as ATP for P2Y1, whereas for P2Y11 ATP
is more potent than ADP (7, 12, 13). UDP selectively activates P2Y6 (7,
58). The unique, naturally occurring agonist of P2X receptors is ATP
(7, 13, 14).
Adenosine and UTP showed no ability to induce phosphorylation of ERK
and p56lck kinase in Jurkat cells at a concentration of 100 µM (Fig. 5A) or
higher (data not shown). These results indicated that the P1 receptor
as well as the P2Y2 and P2Y4 were not involved in the observed
ATP-mediated effects. Next, RT-PCR was performed to test mRNA
expression in the Jurkat cells of five mammalian P2Y receptors (P2Y1,
P2Y2, P2Y4, P2Y6, and P2Y11). We were not able to detect a message
codifying for these receptors (data not shown). More controversial
results were obtained when primers designed to amplify the seven
receptors of the P2X family were utilized. The expression of three
receptors of the P2X family, P2X1, P2X5 (data not shown), and P2X7
(Fig. 5B, left panel), was identified at mRNA
level. Because the fact that only rather high concentrations of ATP (1 mM and above) were required to induce the typical
phosphorylation pattern supports the involvement of the P2X7 receptor
(22, 23, 26), our major interest was focused on the P2X7 subunit.
Therefore, we performed Western blotting experiments with antibodies to
the P2X7 receptor to confirm the presence of the P2X7 protein in the cell lysates (Fig. 5B, right panel).
As a next step, a number of various agonists and antagonists were used
to assess the involvement of each P2X receptor in the ATP-mediated
signaling. Reportedly, suramine antagonizes the effect of ATP on the
P2X purinoreceptors and was shown at concentration of 30 µM to block ATP-induced calcium influx (7, 59).
Pretreatment of Jurkat cells with suramine at a concentration 30 µM for 30 min prior to stimulation with ATP abolished the
phosphorylation of MAP and p56lck kinases (Fig. 5A,
first and third panels). Treatment of Jurkat cells with 100 µM Bz-ATP, which is believed to be a more
potent agonist for the P2X7 receptors than ATP (7, 26), resulted in the
increase of phosphorylation of ERK as well as p56lck kinase at
levels similar to those induced by ATP (Fig. 5A). Further, we employed KN-62, an isoquinoline derivative and inhibitor of CaM
kinase II, which is widely used as a most potent and selective antagonist of the P2X7 receptor (45). In addition, another effective inhibitor of the P2X7 receptor, oATP, was used (46), although data are
available indicating multiple P2X receptor targets for this agent (26).
Pretreatment of Jurkat cells with KN-62 (1 µM) for 5 min
or with oATP (300 µM) for 30 min prior to stimulation with ATP was able to abrogate the activation of the MAP and
p56lck kinases (Fig. 5A, first and
third panels). For loading control, we detected ERK or
p56lck kinase on the same membranes after stripping (Fig.
5A, second and fourth panels).
It has been repeatedly reported that signal transduction through the
P2X7 receptor is associated with Ca2+ influx across the
cellular membrane (5, 32) and may also open a nonselective pore capable
of allowing uptake of low molecular mass hydrophilic solutes (up
to 900 Da), such as Lucifer yellow and ethidium bromide (7, 27).
Therefore, we investigated whether these effects take place in
ATP-stimulated Jurkat cells. ATP at various concentrations (1-3
mM) was not able to induce the uptake of Lucifer yellow
(Fig. 5C) or ethidium bromide (data not shown) by Jurkat
cells. Human macrophages were used as a positive control.
Notwithstanding, as shown in Fig. 5D, both ATP and Bz-ATP were able to induce transmembrane Ca2+ influx, and this
effect was completely abrogated by KN-62, thus providing a further
support for the identification of the P2X7 receptor as responsible for
the ATP-induced effects in Jurkat cells. In addition, as mentioned
above, chelation of extracellular Ca2+ in the culture
medium by EGTA was able to abrogate the ATP-mediated activation of
p56lck and MAP kinases. Taken together, RT-PCR and
pharmacological selectivity data indicate the P2X7 receptor as a most
probable candidate responsible for the induction of the downstream
signaling events after stimulation of Jurkat cells with ATP.
Extracellular ATP Stimulates the Expression of c-Jun and c-Fos
Proteins and Activation of AP-1--
Numerous studies have focused on
the regulation of transcription factors by members of the MAP kinase
family. AP-1 proteins have been identified as substrates of the MAP
kinases (39, 60, 61). The MAP kinase cascade plays a role in the
stimulation of fos gene products, which heterodimerize with
Jun proteins to form more stable AP-1 dimers (60). A major role in
jun induction is played by JNK, which phosphorylates and
enhances the transcriptional activity of two major factors, c-Jun and
ATF2 (60). The observed activation of the p56lck kinase and MAP
kinase pathway in Jurkat cells after ATP treatment strongly invited
further exploration of the changes in the activity of transcription
factors. AP-1 transcription factor usually consists of Jun/Fos
heterodimers (62, 63). To understand whether AP-1 activity is regulated
by the activated MAP kinases upon ATP treatment, we studied first the
changes in the expression of AP-1 components c-Jun and c-Fos, followed
by the evaluation of AP-1 DNA binding activity. Jurkat cells were
stimulated with ATP for different time intervals and assayed for the
expression of c-Jun and c-Fos proteins by SDS-PAGE. As shown in Fig.
6A (top and
middle panels), nonactivated cells express both proteins at
relatively high level. However, stimulation of Jurkat cells by ATP
resulted in a further increase in the expression of both c-Jun and
c-Fos and, likely, also in an enhanced phosphorylation of c-Jun, as
shown by the appearance of shifted up bands of slightly higher
molecular mass (Fig. 6A), although this speculation requires
further proof. The expression of another transcription factor, c-Myc,
which served as a control, remained at the same level (Fig.
6A, bottom panel).
Finally, nuclear extracts from activated cells were analyzed for
AP-1-DNA binding activity using oligonucleotides carrying a consensus
for an AP-1-binding site. ATP induced an enhancement in the AP-1-DNA
binding activity that was already detectable within the first 30 min
and continued to increase, reaching a peak at the third hour of
activation (Fig. 6B). To determine the composition of
ATP-induced AP-1 complex, nuclear extracts from ATP-treated Jurkat
cells were incubated with different antibodies and then assayed for
AP-1 by EMSA. The ability of antibodies against c-Fos and c-Jun to
supershift the band to the higher molecular mass position, as shown in
Fig. 6C, suggested that these two proteins are the part of
activated AP-1 complex. Irrelevant antibodies (anti-cyclin D) had no
effect on the mobility of AP-1. Specificity of the AP-1·DNA complexes
was demonstrated by competition assays where the addition of cold AP-1
oligonucleotides inhibited the formation of AP-1·DNA complex, whereas
mutated AP-1-consensus motif oligonucleotides failed to show any
binding. Taken together, these results indicate that ATP induces the
increase in the expression of c-Jun/c-Fos proteins and enhances
AP-1·DNA binding activity in Jurkat cells.
Extracellular ATP Down-regulates NF- Effects of Extracellular ATP on p56lck-deficient JCaM1
Cells--
To study the possible role of p56lck in
ATP-mediated activation of MAP kinases and up-regulation of AP-1, we
used the JCaM1 cell line, a derivative of Jurkat cells deficient in
p56lck expression: JCaM1 cells reconstituted and stably
expressing p56lck (JCaM1/Lck+) or transfected with a pBP1 mock
plasmid (JCaM/vector+). As shown in Fig.
8A, JCaM1 cells as well as
JCaM/vector+ do not express p56lck kinase. JCaM1/Lck+ cells
have restored expression of p56lck at the level similar to that
of Jurkat cells. These three cell lines exhibit the same pattern of the
P2X7 receptor expression at the mRNA and protein level as Jurkat
cells, as evaluated by RT-PCR and Western blotting, respectively (Fig.
8, B and C). Absence of p56lck in JCaM1
and JCaM/vector+ cells did not affect ATP-mediated Ca2+
influx as shown in Fig. 8D, whereas KN-62, an antagonist of
the P2X7 receptor, was able to completely abrogate this effect in all
cell lines used (data not shown). In addition, ATP was not able to
trigger uptake of Lucifer yellow or ethidium bromide by JCaM1,
JCaM1/Lck+, or JCaM/vector+ cells (data not shown).
Further, the JCaM1 cell lines described above were utilized to study
the importance of p56lck for the ATP-induced intracellular
events. In striking contrast, stimulation with ATP of JCaM1 and
JCaM/vector+ cells did not result in the phosphorylation of ERK and
JNK, increased expression of c-Jun and c-Fos proteins (Fig.
9), and AP-1 activation but induced Ca2+ influx across the plasma membrane (data not shown).
However, JCaM1/Lck+ cells displayed a phosphorylation pattern
resembling that of Jurkat cells, namely the activation of MAP kinases
(Fig. 9) and increased expression of c-Jun and c-Fos (data not shown). Thus, these results strongly suggest that ATP-mediated effects in JCaM1
cells, such as ERK and JNK phosphorylation, increased expression of
c-Jun and c-Fos proteins, and activation of AP-1 but not
Ca2+ influx are dependent on the proper expression and
function of p56lck kinase.
Extracellular ATP Stimulates Proliferation of Jurkat Cells and
Increases Transcription of IL-2--
To understand the biological
significance of the described intracellular events in response to ATP,
we evaluated the consequences of activation of the P2X7 by ATP on
Jurkat cell survival and growth. Given the well documented ability of
ATP to induce cell death via both apoptotic or necrotic mechanisms in
many cell types (28, 31-34), we wanted to test whether such effect
could also be observed in Jurkat and JCaM1 cells. The cells were
cultured in the presence of 3 mM ATP for 24 or 48 h,
and the cell viability was assessed at 24, 48, and 72 h by the
exclusion of propidium iodide. Additionally, the number of the
apoptotic cells was evaluated by flow cytometry analysis using
fluorescein isothiocyanate-conjugated anti-annexin V antibody staining.
As shown in Fig. 10 (A and
B), ATP had no influence on cell survival and the number of
apoptotic cells. About 96% of the cells remained alive after 24, 48, and 72 h. Next, we evaluated the proliferation activity of Jurkat
cells in response to ATP. As shown on Fig. 10C, ATP was able
to stimulate the proliferation of Jurkat cells in a
dose-dependent manner, and the ATP antagonist, oATP, was
able to block the observed proliferation response. This effect,
however, was detectable not only in Jurkat cells but also, although
to a slightly lesser extent, in p56lck-deficient JCaM1 cells,
which do not exhibit the characteristic phosphorylation pattern in
response to extracellular ATP, indicating the proliferation of the
cells in response to the ATP treatment as a more general phenomenon.
This finding strongly invited further exploration of the biological
relevance of the activation of p56lck and MAP kinases in Jurkat
cells. Therefore, as a next step, we tested by semi-quantitative RT-PCR
a panel of various cytokines (e.g. IL-1 Despite the obvious significance and broad distribution of cell
surface receptors for extracellular nucleotides in many cellular systems, including the immune system, intracellular signaling pathways induced by extracellular ATP in lymphocytes are not understood well. This study provides evidence that extracellular ATP is able to
induce activation of p56lck and MAP kinases in Jurkat cells,
followed by up-regulation of AP-1 and down-regulation of NF- The ability of Jurkat cells to proliferate in response to extracellular
ATP, as shown by increased [3H]thymidine incorporation,
confirms the existence of functional purinoreceptors on the cell
surface. Stimulation of DNA synthesis by extracellular nucleotides in a
similar cell model was already documented (67). The apparent lack of a
P2X7-dependent cell death response in this T cell line is
intriguing and deserves further exploration in follow-up experiments.
Interestingly, transfection of P2X7 DNA into lymphoid cells lacking
endogenous P2X7 receptors, K562 and LG14, was able to enhance cell
proliferation in the absence of exogenous growth factors (31).
Furthermore, tumor transformation may lead to an increase in expression
of the P2X7 receptor as well as to the release of a considerably higher
amount of extracellular ATP (68). The possible increase in
proliferation rate, which expression of the P2X7 receptor appears to
bestow upon the cells, may serve as a factor that confers a selective
advantage and enhances their survival. In our experiments, however,
both Jurkat and JCaM1 cells were able to proliferate after ATP
stimulation. Therefore, we made an attempt to identify the biological
significance of the observed signaling pathway in Jurkat cells more
precisely. Reportedly, ATP stimulation may trigger a number of
biologically relevant processes, such as cytokine production, cell
proliferation, or apoptosis (26, 29-34). Semi-quantitative RT-PCR
showed that the up-regulation of IL-2 on a transcriptional level was
already detectable after first 3 h of ATP treatment, continuing to
increase with the time course and reaching its peak after 24 h of
stimulation. In striking contrast, JCaM1 cells exhibited no changes in
the expression pattern of IL-2. Based on these findings, one might conclude that the up-regulated IL-2 transcription in Jurkat cells but
not JCaM1 cells results as a direct consequence of the activation of
the p56lck, MAP kinases and AP-1, thus indicating the
biological significance of the particular signaling pathway involved.
The nucleotide and pharmacological selectivity data strongly suggest
that the ATP-induced downstream intracellular signaling events are
results of activation of the P2X7 receptor. The involvement of P1
receptors was ruled out on the basis of the absence of an effect of
adenosine, which can act as agonist at these receptors. Further, only
millimolar concentrations of extracellular ATP had the capacity to
induce the activation of signaling molecules, whereas ADP, AMP, UTP,
and GTP were not able to trigger such changes in the phosphorylation
pattern (data not shown). Next, the fact that the P2X7 receptor has the
capacity to trigger a long lasting transmembrane Ca2+
influx complies with our observation that ATP-induced intracellular signaling events in Jurkat cells were wholly dependent on cytoplasmic influx of extracellular Ca2+ ions, as shown by the ability
of EGTA, a chelator of divalent cations, to inhibit the protein
phosphorylation via depletion of Ca2+ ions in the culture
medium prior to ATP treatment. The presence of mRNA for the P2X1,
the P2X4, and, particularly, the P2X7 receptors was reported to be
prominent in many immune cells (7, 20, 21). Thus, we tested by RT-PCR
the expression of messages corresponding to the mammalian metabotropic
P2Y receptors and to the ionotropic P2X receptors, with a particular
interest to expression of the P2X7 receptor. RT-PCR confirmed the
presence of mRNA for the P2X7, P2X1, and P2X5 receptors in Jurkat
and JCaM1 cells but failed to detect messages corresponding to the
other receptors of P2X family (P2X2-4 and P2X6) as well as to the
P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptors. The expression of the P2X7
protein in the cells was also confirmed by Western blotting. As a next
step, we evaluated the pharmacological selectivity characteristics to resolve the P2X receptor subtype underlying the intracellular consequences of the ATP action. First, oATP has been extensively utilized as an effective ATP antagonist, which covalently binds to and
inhibits the P2X7 receptor (46), although recent data indicate that
this agent may not differentiate between multiple P2X receptor targets
(26). Therefore, we also used KN-62, an isoquinoline derivative and a
CaM kinase inhibitor, which exhibits a strong
species-dependent sensitivity for human P2X7 and is widely used as a most potent and selective antagonist of the P2X7 receptor (45). The action of KN-62 upon the P2X7 does not involve its inhibitory
properties on calmodulin kinase in short term studies; however,
prolonged exposures to KN-62 require cautious interpretation, because
they may also result in the inhibition of the CaM kinase II and are
thus unsuited for long term studies on cell proliferation. Then, we
used a well known agonist of the P2X7 receptor, Bz-ATP, which is
believed to be more potent than ATP and can mimic its action at lower
concentrations (below 1 mM) (7). Pretreatment of the cells
with oATP, KN-62, or suramin abrogated ATP-induced phosphorylation of
p56lck and MAP kinases, whereas treatment with Bz-ATP mimicked
ATP effects, resulting in the similar phosphorylation pattern. The
ability of Bz-ATP to induce Ca2+ influx in the Jurkat cell
line used in this study appears to contradict a recent report of Vigne
et al. (69), where Bz-ATP (up to 100 µM) had
no effect by itself to increase Ca2+ in Jurkat cells and
served as an antagonist of human and rat P2Y1 receptors. It should,
however, be mentioned that expression of the purinoreceptors may vary
considerably among different clonal variants of the same cell line
maintained under different culture conditions, thus favoring different
selection pressures on the cells that may account for the loss of the
original morphological phenotype because of phenotypic instability, as
was already shown for PC12 cells (54). Therefore, the diverse pattern
of purinoreceptor sensitivity among cell line batches utilized in
different laboratories might contribute to a distinct pattern of
response to extracellular nucleotides (7). The requirement of ATP in a
millimolar range and the ability of submillimolar amounts of Bz-ATP to
mimic its effects taken together with inhibitory properties of KN-62
and oATP are typical for the pharmacological profile of the P2X7
receptor, strongly suggesting a role for it as the sole mediator of the following signaling events in Jurkat cells.
p56lck kinase activity is essential for the initiation of
downstream signaling pathways in T cells (51, 52). The phosphorylation state of Tyr394 stimulates the catalytic activity of
p56lck (52, 53), whereas phosphorylation of Tyr505
helps to stabilize it in a relatively inactive, "closed" biological conformation, resulting in binding of Tyr(P)505 to the Src
homology domain 2 domain of p56lck (51, 52). Nevertheless,
positive regulation of p56lck by phosphorylation at
Tyr394 was shown to be dominant over any inhibition induced
by Tyr505 phosphorylation (53). ATP at high concentrations
and its analog Bz-ATP both were able to induce a rapid and transient
tyrosine phosphorylation of several proteins in Jurkat cells, including p56lck kinase (Figs. 1 and 5), and this effect was dependent on
the presence of calcium in the extracellular medium. Through the use of
phosphorylation state-specific antibodies, we have shown that p56lck was phosphorylated at Tyr394 but not at
Tyr505, resulting presumably in kinase activation. To prove
this further, we performed a kinase assay that confirmed catalytic
activation of p56lck kinase by specific phosphorylation of
TCR The p56lck SH3 domain was shown to play an important role in
the activation of MEK and ERK following TCR stimulation (38). In
addition, p56lck kinase was recently shown to be required for
the ceramide-induced and HIV-tat protein-induced activation of MEK and
JNK, AP-1, and NF- A plethora of physiological and pathological stimuli is able to induce
and activate a group of DNA-binding proteins that form the AP-1
complex, which binds to AP-1 regulatory elements in the promoter and
enhancer regions of numerous mammalian genes, including IL-2 (65, 66).
In unstimulated T cells, AP-1 expression is low or undetectable, with a
rapid induction of AP-1 activity after T cell stimulation, such as
TCR/CD3 binding and action of cytokines or hormones (71-73). The
ATP-mediated induction of c-Jun and c-Fos expression, followed by the
up-regulation of DNA binding activity of AP-1 may mirror, at least in
part, the initial steps in T cell activation, which are usually
orchestrated through the recruitment of several transcriptional
factors, such as AP-1, nuclear factor of activated T cells
(NFAT), and NF- Another important inducible transcription factor that plays a pivotal
role in many cellular responses to environmental changes is NF- Taken together, our results demonstrate that ATP induces
Ca2+-dependent activation of p56lck
tyrosine kinase, which is prerequisite for the activation of ERK and
JNK MAP kinases, up-regulates transcriptional factor AP-1, and
stimulates IL-2 transcription, while down-regulating DNA binding activity of NF-B was reduced. ATP failed to
stimulate the phosphorylation of ERK and c-Jun N-terminal kinase and
activation of AP-1 in the p56lck-deficient isogenic T cell line
JCaM1, suggesting a critical role for p56lck kinase in
downstream signaling. Regarding the biological significance of the
ATP-induced signaling events we show that although extracellular ATP
was able to stimulate proliferation of both Jurkat and JCaM1 cells, an
increase in interleukin-2 transcription was observed only in Jurkat
cells. The nucleotide selectivity and pharmacological profile data
supported the evidence that the ATP-induced effects in Jurkat cells
were mediated through the P2X7 receptor. Taken together, these results
demonstrate the ability of extracellular ATP to activate multiple
downstream signaling events in a human T-lymphoblastoid cell line.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 and
IL-18 in human monocytes (29, 30), altered cytokine production in mice
lacking this receptor (31), stimulation of JNK activity and induction
of apoptosis in murine macrophages (26), inhibition of osteoclastic
resorption (32), and activation of transcription factor NF-
B (33)
and nuclear factor of activated T cells (NFAT) (34) in
microglial cell lines. Despite the accumulating evidence for an
important function of the P2X7 receptor in many cellular systems,
intracellular signaling events underlying the biological processes upon
the P2X7 stimulation in immune cells are still obscure.
B
transcription factor was reduced. The observed effects were wholly
dependent on the extracellular Ca2+ influx and, in regard
to the pharmacological profile and expression pattern, are likely to be
exclusively mediated by the P2X7 receptor. Finally, the experiments
aimed to characterize the biological significance of the signaling
pathway involved showed that ATP was able to stimulate proliferation of
Jurkat cells and to induce an increase in transcription of IL-2. Thus,
the data presented in this study demonstrate the ability of
extracellular ATP to elicit diverse cellular responses in a human
T-lymphoblastoid cell line.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B p50 (C-19), p65 (A), c-Rel (N-466), cyclin D (R-124),
Fos (4), Jun (N), Myc (C-19), P2X7 (L-20) were purchased from Santa
Cruz (Santa Cruz, CA); anti-pLck (pY505) and anti-pSrc (pY416) were
purchased from BioSource Int. (Camarillo, CA).
80 °C until electrophoresis.
-32P]ATP (3000 Ci/mmol; Amersham Biosciences), 10 µM ATP, and 1 µg of GST-
TCR fusion protein
(p56lck substrate, generously provided by Dr. D. Straus) for 15 min at 37 °C. The reaction was stopped by adding 20 µl of 4×
sample buffer. The samples were boiled for 5 min, and the proteins were
resolved in 10% SDS-PAGE. The phosphotyrosine-containing
proteins were detected by autoradiography.
-actin (GenBankTM
accession number NM 001101): sense, 5'-GTGGGGCGCCCCAGGCACCA-3', and
antisense, 5'-CTCCTTAATGTCACGCACGATTTC-3'. All of the primers were purchased from TIB Molbiol (Berlin, Germany). The samples were
amplified in a DNA Thermocycler (Eppendorf, Hamburg, Germany) for 35 cycles. Each cycle consisted of denaturation at 94 °C for 30 s,
annealing at 60 °C for 1 min, and extension at 72 °C for 1 min.
Aliquots of PCR products were electrophoresed on 1.5% agarose gel and
visualized by ethidium bromide staining. To evaluate mRNA expression semi-quantitatively, in addition to the PCR product from 35 cycles, 15 µl of the PCR product from the 25 cycles and the 30 cycles
was run simultaneously.
-Actin message was used to normalize the
cDNA amount to be used. A mock PCR (without cDNA) was included
to exclude contamination in all experiments.
B oligonucleotides from the HIV long terminal
repeat,
5'-TTGTAACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3' or with AP-1 oligonucleotides, 5'-CGCTTGATGACTCAGCCGGAA-3'
and 3'-GCGAACTACTGAGTCGGCCTT-5' (underlined regions
are consensus NF-
B or AP-1 sites). A binding reaction was performed
in 20 µl of reaction buffer (20 mM Tris-Cl, pH 7.9, 20%
glycerol, 50 mM KCl, 1 mM dithiothreitol, and
2.5 mM MgCl2) containing 4 µg of nuclear
proteins, 2 µg of poly(dI-dC), and 1 ng of end-labeled DNA
(10,000-30,000) at room temperature, loaded on 4% polyacrylamide gel,
and run in 0.5× TBE buffer (89 mM Tris, 89 mM
boric acid, pH 8.3). The gels were dried in a gel dryer and exposed to
an x-ray film. A double-stranded mutated oligonucleotides were used to
prove the specificity of binding of NF-
B or AP-1 to the DNA. The
specificity was also proven by competition with the unlabeled ("cold") oligonucleotides. To characterize NF-
B·DNA and
AP-1·DNA complexes, supershifting assay was performed using anti-p65,
anti-p50, anti-c-Rel antibodies, and anti-c-Fos/c-Jun antibodies, respectively.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
ATP-induced tyrosine phosphorylation of
intracellular proteins. Jurkat cells were serum-starved for 3 h and incubated at 37 °C in the absence (control) or
presence of 3 mM ATP for different time intervals, as
indicated. The proteins were precipitated using biotinylated
anti-Tyr(P) antibodies (RC20B). A, phosphorylated proteins
were detected in immunoblotting using RC20H antibodies. B,
the same blot was stripped with 62.5 mM Tris-HCl buffer, pH
6.7, containing 2% SDS and 100 mM -mercaptoethanol
overnight at 4 °C and reprobed with anti-Lck antibodies.
C, lysates from ATP-activated Jurkat cells were precipitated
with anti-Lck antibodies, and immunoblotting with anti-Tyr(P)
antibodies was performed. D, detection of p56lck
after immunoprecipitation as a control of loading. The positions of
phosphorylated proteins are indicated on the left, and
phosphorylated p56lck is indicated on the right. The
picture presents one of three independent experiments with same
results. IP, immunoprecipitation; WB, Western
blot.
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Fig. 2.
ATP stimulates phosphorylation of
Tyr394 but not Tyr505 of p56lck and
increases its kinase activity. Serum-starved Jurkat cells were
stimulated with 3 mM ATP for the indicated time periods at
37 °C. A, p56lck kinase was precipitated from
lysates and phosphorylation of Y394 (top panel), and Y505
(middle panel) was detected using anti-Src (pY416) and
anti-Lck (pY505) antibodies, respectively. The bottom panel
shows the loading control. B, in vitro kinase
assay of p56lck immunoprecipitates from ATP-activated Jurkat
cells. Immunocomplexes were incubated with [ -32P]ATP
and GST-
TCR fusion protein as an exogenous substrate (upper
panel). A fraction of each immunoprecipitate was immunoblotted
with anti-Lck antibodies to prove the equal loading (lower
panel). C, Jurkat cells were treated with 3 mM ATP for 5 min, and the activation of p56lck was
assessed in absence or presence of calcium chelator EGTA. The chelation
of Ca2+ ions in extracellular medium was performed by
adding to the medium of 2 mM EGTA. The results are
representative of three independent experiments. IP,
immunoprecipitation; WB, Western blot.
-32P]ATP and an exogenous substrate consisting
of the cytosolic domain of the
-chain of the TCR fused to GST (38).
As it is shown in Fig. 2B, ATP activates the kinase activity
of p56lck within 5-15 min of treatment. To investigate whether
Ca2+ was essential for ATP-induced p56lck
phosphorylation, Jurkat cells were stimulated with ATP in the presence
of the Ca2+ chelator EGTA. As shown in Fig. 2C,
pretreatment of cells with 2 mM EGTA completely prevented
p56lck phosphorylation, indicating that the activation of
p56lck required extracellular Ca2+. Thus,
extracellular ATP mediates Ca2+-dependent
activation of p56lck in Jurkat cells by inducing
phosphorylation at Tyr394 and increases the activity of the kinase.
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Fig. 3.
ATP induces phosphorylation and activation of
ERK and JNK but not p38 kinase. Serum-starved Jurkat cells were
stimulated at 37 °C with 3 mM ATP for the indicated
times and lysed. Then equal amounts of the proteins were loaded onto
10% gel, and SDS-PAGE was performed. Phosphorylation of ERK 1/2,
JNK1/2, and p38 kinases at specific sites was detected using
phosphospecific antibodies. The positions of phosphorylated kinases are
indicated on the right. The equal amounts of ERK, JNK, and
p38 are shown as controls for loading. The data shown are
representative of three separate experiments with similar
results.
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Fig. 4.
Effect of PD098059, calmidazolium, and KN-93
on phosphorylation of ERK and p56lck kinase. A,
serum-starved Jurkat cells were incubated at 37 °C with 50 µM of MEK inhibitor, PD98059, for 90 min prior to the ATP
treatment and stimulated thereafter for different time intervals with 3 mM ATP. The cells were analyzed for ERK phosphorylation
using specific anti-pERK antibodies (top panel).
Phosphorylation of p56lck was evaluated by immunoprecipitation
with anti-Lck antibodies and Western blotting with anti-Src(pY416)
antibodies (third panel). Equal loading of ERK and
precipitation of p56lck are shown at second and
fourth panels, respectively. B, Jurkat cells were
serum-starved and treated at 37 °C with 50 mM
calmidazolium or 1 µM KN-93 for 30 min prior to the
activation with 3 mM ATP (5 and 15 min). Then the membranes
were probed with activation state-specific anti-pERK antibodies to
determine the phosphorylation of ERK, whereas the activation of
p56lck was detected by immunoprecipitation with anti-Lck
antibodies and Western blotting with anti-Src(pY416) antibodies
(third panel). Pretreatment of cells with Me2SO
(1:100) or KN-92 (1 µM) and activation with 500 nM of calcium ionophore A 23187 were used as controls.
Equal loading of ERK and precipitation of p56lck are shown in
the second and fourth panels. IP,
immunoprecipitation; WB, Western blot.
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Fig. 5.
ATP induces the phosphorylation of ERK,
p56lck kinase and Ca2+ influx in Jurkat cells
through the P2X7 receptors but does not influence cell membrane
permeability. A, Jurkat cells were serum-starved for 3 h and stimulated for 15 min at 37 °C with 3 mM ATP or
with appropriate purinoreceptor agonists (Bz-ATP, adenosine, or UTP) at
concentration of 100 µM. Additionally, the cells were
incubated for 30 min with 30 µM suramine, for 15 min with
300 µM oATP, or for 5 min with 1 µM KN-62
prior to stimulation with 3 mM of ATP for the next 15 min.
Then the cell lysates were analyzed for the phosphorylation of ERK and
p56lck kinase. B, total RNA extracted from Jurkat
cells was reverse transcribed into cDNA, and PCR using primers to the P2X7 receptor was performed (left panel). The
amplified products from this reaction were analyzed by 1.5% agarose
gel electrophoresis. A mock PCR ( DNA) was included as a
negative control. Expression of the P2X7 receptor was analyzed in
Jurkat cell lysates using specific anti-P2X7 antibodies (right
panel). The position of the P2X7 receptor is indicated on the
right. C, membrane permeabilization was analyzed
in Jurkat cells by fluorescence microscopy (right panels).
The cells were incubated in the absence (control) or presence of 3 mM ATP for 15 min in a buffer containing 1 mg/ml Lucifer
yellow, washed, and examined under phase contrast (left
panels) or fluorescent light using a fluorescent filter
(right panels). Membrane permeability induced by ATP in
human macrophages is shown as a control. The cells were observed with a
40× objective. D, ATP- and Bz-ATP-mediated Ca2+
influx is inhibited by KN-62 in Jurkat cells. Ca2+ influx
in Jurkat cells was measured as described under "Materials and
Methods" after stimulation with 3 mM ATP or 100 µM Bz-ATP in control cells (left graphs) or
after treatment with 1 µM KN-62 for 5 min (right
graphs). The data shown are representative of three independent
experiments with similar results. IP,
immunoprecipitation.
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Fig. 6.
ATP treatment stimulates an increase in the
expression of c-Jun and c-Fos and enhances AP-1 DNA binding
activity. A, serum-starved Jurkat cells were stimulated
with 3 mM ATP and lysed, and proteins were analyzed by
Western blotting. Then the membranes were probed with anti-c-Jun (p39)
and anti-c-Fos (p60) antibodies. Expression of c-Myc (p65) was assayed
at the same time for comparison and loading control. B,
nuclear extracts from the stimulated cells were incubated with
double-stranded oligonucleotides with an AP-1 consensus site, followed
by electrophoretic mobility shift assay (EMSA) as described under
"Materials and Methods." The position of AP-1·DNA binding complex
is indicated on the right. C, nuclear extracts
were prepared from treated for 3 h with ATP (3 mM)
Jurkat cells and incubated with c-Jun, c-Fos, JunD, and irrelevant
anti-cyclin D1 antibodies for 15 min and then assayed for AP-1 as
described under "Materials and Methods." Competition with unlabeled
intact or mutated AP-1 probes shows the specificity of binding.
B Transcription
Factor--
The NF-
B family of transcription factors controls the
expression of numerous genes involved in T cell function and can be activated in response to a broad variety of stimuli. Many agents that
activate NF-
B also activate AP-1 transcription factor (57, 64).
Therefore, as a next step, we wanted to study the effect of ATP on
NF-
B activity. Jurkat cells were treated for different time
intervals with ATP, and the nuclear extracts were examined for NF-
B
DNA binding activity by EMSA. Fig.
7A shows that ATP transiently
inhibits NF-
B binding activity within 1-2 h of treatment. To prove
that the retarded band visualized by EMSA is NF-
B, we incubated
nuclear extracts with antibodies against p50 (NF-
BI), p65 (Rel A),
or c-Rel and then performed EMSA. Antibodies against p50 and p65 of
NF-
B supershifted the band to the higher molecular mass position
(Fig. 7B), suggesting that ATP-activated complex consists of
these two subunits. Either anti-c-Rel or irrelevant antibodies
(anti-cyclin D) had no effect on the mobility of NF-
B. Specificity
of binding was proved using EMSA with cold NF-
B oligonucleotides. Incubation of nuclear extracts with unlabeled oligonucleotides completely blocked NF-
B·DNA complex formation (Fig.
7B), and mutated NF-
B consensus motif oligonucleotides
failed to bind NF-
B. Therefore, extracellular ATP is able to
down-regulate the NF-
B transcription factor in Jurkat cells.
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Fig. 7.
Effect of ATP on DNA binding activity of
NF- B. A, Jurkat cells were
treated with ATP for different time intervals as shown. Then nuclear
extracts were incubated for 1 h with 32P-labeled
double-stranded oligonucleotides with consensus sequence for NF-
B
and evaluated by EMSA as described under "Materials and Methods."
The position of the NF-
B·DNA complex is indicated on the
right. B, the nuclear extracts from nonactivated
Jurkat cells were incubated for 15 min with indicated antibodies and
then for an additional 1 h with NF-
B consensus
oligonucleotides, and the complexes were analyzed by EMSA. Controls
with competing unlabeled intact or mutated probes for NF-
B confirm
specificity of binding. The data shown are representative of three
independent experiments with similar results.
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Fig. 8.
Expression of p56lck kinase and the
P2X7 receptor and Ca2+ influx in response to ATP in Jurkat
and JCaM1 cells. A, expression of Lck protein was
detected by Western blotting using anti-Lck antibodies in Jurkat cells,
JCaM1 cells deficient for p56lck expression, JCaM1 cells
reconstituted for p56lck expression (JCaM1/Lck+), and JCaM1
cells transfected with a mock plasmid (JCaM/vector+). B,
expression of the P2X7 receptor was detected by RT-PCR in all of the
cell lines used. C, expression of the P2X7 protein was
analyzed in cell lysates using anti-P2X7 antibodies. D,
cells were stimulated with 3 mM of ATP and Ca2+
influx was measured as described under "Materials and Methods." A
representative of three independent experiments is shown.
View larger version (63K):
[in a new window]
Fig. 9.
ATP failed to stimulate phosphorylation of
ERK and JNK and expression of c-Jun and c-Fos in
p56lck-deficient JCaM1 cells. A, Jurkat, JCaM1,
JCaM1/Lck+ and JCaM/vector+ cells were serum-starved for 3 h and
activated with 3 mM ATP for 5 and 15 min at 37 °C. The
protein lysates were analyzed for ERK and JNK phosphorylation. The
bottom panel represents control of loading. B,
Jurkat and JCaM1 cells were activated for different time intervals, and
expression of c-Jun and c-Fos was detected by SDS-PAGE. Expression of
c-Myc is shown for loading control (bottom panel). The
position of specific proteins is indicated on the right. The
data shown represent three separate experiments, all of which yielded
highly comparable results.
, IL-2, IL-4,
IL-7, IL-15, and IL-18) for possible changes in the level of
transcription. Among these, only transcript codifying for IL-2 was
clearly up-regulated (Fig. 10D, upper panel). The
MAP kinase pathway and activation of AP-1 transcription factor was
reported by several groups to be required for IL-2 production (65, 66).
Unstimulated Jurkat and JCaM1 cells showed a weak transcription of IL-2
cytokine. However, stimulation with 3 mM ATP induced an
increase in the amount of IL-2 transcript in Jurkat cells after only
3 h of ATP action, reaching its peak within 24 h of
stimulation, whereas in JCaM1 cells IL-2 transcription remained at the
same level throughout the time course of the experiment. Taken
together, these results indicate that extracellular ATP has ability to
induce proliferation of both Jurkat and JCaM1 cells and, presumably
through the activation of p56lck, MAP kinases and AP-1, to
selectively up-regulate IL-2 at transcriptional level in Jurkat cells
alone.
View larger version (39K):
[in a new window]
Fig. 10.
ATP stimulates proliferation of Jurkat and
JCaM1 cells and increases transcription of IL-2 in Jurkat cells.
Jurkat and JCaM1 cells were incubated at concentration of 1 × 105 cells/ml in absence or presence of 3 mM ATP
for indicated time intervals. Cell viability (A) was
analyzed by propidium iodide exclusion and fluorescence-activated cell
sorter analysis after 24, 48, and 72 h, and percentage of
apoptotic cells (B) was determined by annexin-fluorescein
isothiocyanate staining after 24 and 48 h. C, Jurkat
and JCaM1 cells were seeded in triplicates (105 cells/well)
and incubated for 24 h with 3 mM ATP. The samples
treated with oATP (300 µM) were cultured in the presence
of this inhibitor throughout the experiment. Then
[3H]thymidine was added for additional 12 h. *,
p < 0.05 versus medium and ATP + oATP.
B, Jurkat and JCaM1 cells were cultured for 3 or 24 h
in the presence of 3 mM of extracellular ATP. Then mRNA
was extracted, reverse-transcribed to cDNA, and amplified with
respective primers (human IL-2, upper panels) for 35 cycles.
KN-62 (1 µM) was used to inhibit the action of ATP.
-Actin message was used to equalize the amount of cDNA used
(lower panels). The amplified PCR products were analyzed by
1.5% agarose gel electrophoresis. For semi-quantitative analysis, in
addition to 35 cycles, 15-ml aliquots of the PCR product from 25 and 30 cycles were also evaluated. The picture shows the amplified bands after
35 cycles. A mock PCR (no cDNA) was included as a negative control.
The data represent three separate experiments with comparable
results.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B
transcription factors. By using p56lck-deficient cells, we
demonstrate that p56lck is required for the ATP-mediated
downstream signaling, whereas the transfection with p56lck gene
reconstituted the ATP-induced cellular responses. To our knowledge,
this is the first report to indicate that p56lck kinase is
required for ATP-mediated activation of AP-1, ERK and JNK, stimulation
of IL-2, and down-regulation of NF-
B in Jurkat cells.
-chain-GST fusion protein as an endogenous substrate for
p56lck.
B transcription factors in Jurkat cells (59, 70).
Importantly, calcium ionophores can trigger activation of
p56lck and ERK1/2 in Jurkat cells via calmodulin and a CaM
kinase-dependent mechanism (44). Furthermore, one signaling
pathway that is required for IL-2 production is MAP kinase cascade
through the action of the transcription factor AP-1 (65, 66). The
involvement of p56lck in TCR signaling appears to be complex,
indicating its selective requirement for the activation of downstream
signaling pathways, such as MAP kinase pathway, despite the activation
of ZAP-70 and phosphatidylinositol in a p56lck-deficient JCaM1
cell line (38). To examine the role of p56lck in the
ATP-induced downstream signaling more precisely, we also employed JCaM1
cells, a genetic variant of Jurkat cell line, deficient in
p56lck protein expression because of the deletion of exon 7 (36, 38). ATP failed to stimulate activation of MAP kinases ERK1/2 and
JNK1/2 and to induce an increase in the DNA binding activity of AP-1 in
these cells, whereas transfection with the gene coding for p56lck reconstituted the ATP-induced cellular responses. Thus,
the selective disruption of the MAP kinase pathway in
p56lck-deficient cells demonstrates that there exists an
additional requirement of this kinase for the activation of downstream
signaling events and suggests an upstream location for p56lck
in the ATP signaling pathway. The activation of p56lck kinase
upon the ATP action does not seem to depend on or be preceded by the
activation of other mediator of Ca2+ signaling, Pyk2 (data
not shown), nor does it require the involvement of calmodulin and CaM
kinase, as shown by the inability of both calmidazolium, a calmodulin
antagonist, and CaM kinase inhibitor KN-93 to abrogate the activation
of p56lck and ERK, although CaM kinase appears to participate,
at least partially, in the observed activation of ERK. Furthermore, a
specific inhibitor of MEK, PD098059, had capacity to abrogate
phosphorylation of ERK in response to ATP, while not affecting
activation of p56lck kinase. The fact that extracellular ATP,
acting over the P2X7 receptor, is able to stimulate
p56lck-dependent phosphorylation and activation of
ERK and JNK, but not p38 kinase, demonstrates a novel pathway by which
this agent can modulate the intracellular responses on various levels.
The precise molecular mechanisms underlying the ability of ATP to trigger the activation of p56lck and MAP kinases through the
P2X7 receptor remain unknown. Although an increase in the intracellular
level of Ca2+ appears to be critical for the subsequent
signaling events, the inability of the P2X7 to mediate activation of
the MAP kinases in the absence of p56lck in JCaM1 cells
unambiguously indicates that a simple rise in the level of
intracellular Ca2+ ions is not enough. Although MEK seems
to contribute to the observed activation of ERK, it is not clear yet
how p56lck kinase might couple to MEK and, in general, mediate
its effects on activation of the MAP kinases and AP-1 in response to
ATP, and future studies are required to address this question in more detail.
B (73). AP-1 probe and nuclear proteins were
able to form complexes within 30 min from the action of ATP and were
still present after 3 h. The supershifting experiments showed that
c-Jun and c-Fos proteins are the constituents of the activated AP-1
transcription complex. As already mentioned above, the MAP kinase
cascade is crucial for AP-1 induction and IL-2 gene expression in T
cells (65, 66), because it regulates tightly at the transcriptional and
post-translational levels of AP-1 activity, increasing its stability
and binding activity through phosphorylation (73, 74).
B.
The factor is ubiquitously found as an inactive complex in the
cytoplasm bound to its inhibitory subunit I
B and plays an important
role in the cell growth, differentiation, development, apoptosis,
inflammation, and immune responses (75, 76). NF-
B is also able to
function in concert with other transcription factors, such as AP-1
(57). In contrast to the activation of AP-1, we detected a
simultaneous, although slightly accelerated decrease in DNA binding
activity of NF-
B p65 (Rel A) and p50 proteins, as evidenced by the
supershift experiments, within the first hour of the ATP action. The
level of NF-
B proteins slowly returned nearly to basal after 3 h. It should be mentioned that we did not observe an increase in the
expression of I
B
upon ATP
treatment.2 The biological
relevance of such opposite responses as the activation of AP-1 and
down-regulation of NF-
B is not clear, and future experiments must be
aimed to shed light on this issue.
B. Thus, the intracellular events occurring in Jurkat
cells in response to extracellular ATP may represent a heretofore
unappreciated mechanism of its ability to modulate cellular processes
within the immune system.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. David Straus for the generous
gift of parental and transfected JCaM1 cell lines and for GST-TCR
and Martina Hein for excellent technical assistance.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
¶ To whom correspondence should be addressed: Dept. of Immunology and Cellular Biology, Research Center Borstel, Parkallee 22, D-23845 Borstel, Germany. E-mail: ebulanova@fz-borstel.de.
Published, JBC Papers in Press, November 6, 2002, DOI 10.1074/jbc.M206383200
2 E. Bulanova and V. Budagian, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
IL, interleukin;
CaM
kinase, Ca2+/calmodulin-dependent protein
kinase;
MAP, mitogen-activated protein;
ERK, extracellular-signal
regulated kinase;
EMSA, electromobility shift assay;
JNK, c-Jun
N-terminal kinase;
Bz-ATP, 3-O-(4'-benzoyl)-benzoyl-ATP;
NF-B, nuclear factor-
B;
oATP, oxidized ATP;
MEK, MAP kinase
kinase;
RT, reverse transcription;
HIV, human immunodeficiency
virus;
GST, glutathione S-transferase;
TCR, T cell
receptor.
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