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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 10 (May 15), 1998:
pp. 3566-3573
Activation of Stat-3 Is Involved in the Induction of Apoptosis After
Ligation of Major Histocompatibility Complex Class I
Molecules on Human Jurkat T Cells
By
Søren Skov,
Mette Nielsen,
Søren Bregenholt,
Niels Ødum, and
Mogens H. Claesson
From the Laboratory of Experimental Immunology, Department of
Medical Anatomy, the Panum Institute, University of
Copenhagen, Copenhagen, Denmark.
 |
ABSTRACT |
Activation of Janus tyrosine kinases (Jak) and Signal transducers
and activators of transcription (Stat) after ligation of major
histocompatibility complex class I (MHC-I) was explored in
Jurkat T cells. Cross-linking of MHC-I mediated tyrosine
phosphorylation of Tyk2, but not Jak1, Jak2, and Jak3. In addition, the
transcription factor Stat-3 was tyrosine phosphorylated in the
cytoplasma and subsequently translocated to the cell nucleus. Data
obtained by electrophoretic mobility shift assay suggested that the
activated Stat-3 protein associates with the human serum-inducible
element (hSIE) DNA-probe derived from the interferon-
activated site (GAS) in the c-fos promoter, a common DNA sequence for
Stat protein binding. An association between hSIE and Stat-3 after
MHC-I ligation was directly demonstrated by precipitating Stat-3 from
nuclear extracts with biotinylated hSIE probe and avidin-coupled
agarose. To investigate the function of the activated Stat-3, Jurkat T cells were transiently transfected with a Stat-3 isoform lacking the
transactivating domain. This dominant-negative acting Stat-3 isoform
significantly inhibited apoptosis induced by ligation of MHC-I. In
conclusion, our data suggest the involvement of the Jak/Stat signal
pathway in MHC-I-induced signal transduction in T cells.
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INTRODUCTION |
THE MAJOR histocompatibility
complex class I (MHC-I) complex is known for its capacity to present
peptide derived from intracellular proteins to cytotoxic T
lymphocytes.1 Recently, there has been a growing interest
in defining the intracellular signals and their consequences for cell
activation after ligation of MHC-I molecules. Thus, it is now well
established that ligation of MHC-I molecules expressed on T lymphocytes
are critically involved in regulation of T-lymphocyte
activation.2-6 In an effort to clarify the intracellular signal pathway(s) that operates after MHC-I ligation, we have previously shown that MHC-I cross-linking of human T lymphocytes induces tyrosine kinase and PLC-1 activity.7 It has been
demonstrated by several groups that surface expression of the TCR/CD3
molecule is essential for MHC-I-induced signal
transduction.7-9 In line with this, we have recently shown
that the MHC-I molecule directly uses parts of the TCR/CD3 signal
transduction machinery for signal transduction, but that MHC-I
cross-linking leads to an alternative TCR/CD3  -chain phosphorylation
resulting in an altered activation of the ZAP70 tyrosine kinase and
induction of apoptosis.10 Deletion of all but the four
proximal amino acids from the intracellular domain of the MHC-I
molecule does not alter its signal transduction capabilities,11,12 strongly suggesting that ligated MHC-I
molecules, to transmit a signal, may associate with other signal
transducing transmembrane molecules. Interestingly, it has been
demonstrated that MHC-I molecules can associate with receptores for
interleukin-2 (IL-2) and IL-4 as well as insulin and glucagon
receptors.13-17
Ligand binding of many different cytokine receptors induces Jak
tyrosine kinase activity and Jak's become noncovalently associated with the actual cytokine receptors. The Jak kinase family consists of
Tyk2, Jak1, Jak2, and Jak3, which are characterized by the presence of
two C-terminal kinase-related domains and the absence of SH2
domains.18,19 The Stat proteins are key substrates for the
Jak kinases. Stat proteins are SH2-containing cytoplasmic proteins,
which associate with the ligand activated cytokine-receptor/Jak kinase
complex via the SH2 domain of the Stat proteins.19 Upon tyrosine phosphorylation, Stat proteins are released from the complex
and form SH2 domain mediated homodimeric or heterodimeric complexes.19,20 These complexes are then translocated to
the cell nucleus, where they regulate gene transcription through
interaction with specific DNA sequences, most of which are related to
the interferon- (IFN-) activated site (GAS), a regulatory element in the IFN- inducible genes.21 Presently, six members of
the Stat family are identified and some of these exist in different isoforms.22 Each Stat protein functions in specific
cytokine-receptor signal pathways. Stat-3, for example, is activated by
various receptors such as the IL-2 receptor,23 IL-3
receptor,24 IL-5 receptor,25 IL-6
receptor,26 IL-9 receptor,27 IL-10
receptor,28 colony-stimulating factor-1,29
epidermal growth factor (EGF) receptor,30,31
and the thrombopoitin receptor.32 Evidence suggests that
the specificity of Stat phosphorylation is not due to the specificity
of Jak activation. Rather, it is hypothesized that the receptor complex
determines which Stat proteins will be accessible to
phosphorylation.19 The aim of the current study was to
investigate the involvement of the Jak/Stat signal pathway after MHC-I
ligation of human Jurkat T cells.
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MATERIALS AND METHODS |
Antibodies (Abs) and reagents.
Purified antihuman  2 microglobulin
(anti-2m) Ab from rabbit serum (DAKO A072; DAKO,
Roskilde, Denmark). Purified control Ig from rabbit serum (DAKO X903).
F101.01 monoclonal antibody (MoAb), which recognizes a conformational
determinant on TCR33 was used as an ascites dilution. We
used anti-CD3 (UCHT1) MoAb (DAKO M835); antiphosphotyrosine MoAb, IgG2b
(UBI #05-321); purified antihuman Tyk2 from rabbit serum (UBI #06-275);
purified antihuman Jak1 from rabbit serum (UBI #06-272); purified
antihuman Jak2 from rabbit serum (UBI #06-255); purified antihuman Jak3
from rabbit serum (UBI #06-428); antihuman Stat1, MoAb, IgG2b (Affinity #S21120; Mamhead, Exeter, UK); antihuman Stat2, MoAb, IgG2a (Affinity #S21220); antihuman Stat-3, MoAb, IgG1 (Affinity #S21320); antihuman Stat4, MoAb, IgG1 (Affinity #S21420); antihuman Stat5, MoAb, IgG2b (Affinity #S21520); antihuman Stat6, MoAb, IgG2b (Affinity #S25420); antihuman ISGF3, MoAb, IgG1 (Affinity #129320);
peroxidase-conjugated antimouse Ig from rabbit serum (DAKO P260); and
purified peroxidase-conjugated antirabbit Ig from swine serum (DAKO
Z196). Abs used for cell exposure were dialyzed against
phosphate-buffered saline (PBS) before use and used at the indicated
concentrations. Abs for immunoprecipitation were used according to the
manufacturer's description. Biotin-conjugated Abs were prepared by
reacting Ab with biotinsuccinimide (Sigma B-2643; Sigma,
St Louis, MO), as described in Odum et al.34 Avidin (Sigma
A9275) was used to cross-link biotin-conjugated Ab:
natrium-orthovanadate (Na3VO4; Sigma S6508);
protein A sepharose CL-4B (Pharmacia, Uppsala, Sweden); avidin-coupled
agarose (KemEnTec, Copenhagen, Denmark); Ripa buffer (10 mmol/L
Tris/HCl, pH 7.5, 1% NP-40, 0.25% deoxycholate wt/vol, 2 mmol/L EDTA,
10 mmol/L orthovanadate); lysis buffer for nuclear and cytoplasmic
purification (10 mmol/L Tris/HCl, pH 7.5, 0.5% Triton X-100, 2 mmol/L
EDTA, 10 mmol/L orthovanadate); buffer for nuclear purification (10 mmol/L KH2PO4, pH 6.8, 2.2 mol/L sucrose, 1 mmol/L MgCl2, 150 mmol/L NaCl, 10 mmol/L orthovanadate);
buffer for cytoplasmic purification (10 mmol/L Tris/HCl, pH 7.5, 0.25 mol/L sucrose, 1 mmol/L MgCl2, 150 mmol/L NaCl, 10 mmol/L
orthovanadate); electrophoretic mobility shift assay (EMSA) lysis
buffer I (40 mmol/L Tris/HCl, pH 7.0, 2 mmol/L EGTA, 5 mmol/L
MgCl2, 1 mmol/L phenylmethylsufonyl fluoride
[PMSF], 0.1 mmol/L Na3VO4, 10 mmol/L NaF, 0.1 mmol/L NH4Molybdate, 10 µmol/L Pepstatin,
10 mmol/L -glycerophosphate, 5 mmol/L dithiothreitol [DTT], 15 mmol/L p-nitrophenylphosphate, 10 µmol/L Leupeptin); and
EMSA lysis buffer II (lysis buffer I supplemented with 50 mmol/L KCl,
300 mmol/L NaCl, 1.5% Ficoll). All buffers, except EMSA lysis buffers,
were supplemented with a protease inhibitor cocktail according to the
manufactures description (Boehringer Mannheim Gmbh #1697498; Boehringer
Mannheim, Mannheim, Germany).
Cells.
Jurkat cell J76.25 was kindly provided by Dr C. Geisler (Institute for
Medical Microbiology and Immunology, University of Copenhagen,
Copenhagen, Denmark). Cells were grown in RPMI 1640 with 10%
heat-inactivated fetal calf serum (FCS), fresh L-glutamine, and
antibiotics. Cells were continuously tested to be mycoplasma free.
Cell stimulation.
Cells were preincubated with saturating amounts of biotinylated
anti-2m Ab or biotinylated control rabbit Ig (both 1:10
of batch concentration) using 10 µL antibody/106 cells in
a final volume of 100 µL for 10 minutes at room temperature. Subsequently, cells were washed in PBS (37°C) and cross-linked with
avidin (20 µg/106 cells) or UCHT-1 Ab (2.5 µL/106 cells) in a final volume of 100 µL at 37°C for
various periods of time.
Immunoprecipitation.
Cells (3 × 107) were treated as described above, and
the pellet was lysed in 1 mL ripa buffer and precleared several times with sepharose-coupled protein A (50% wt/vol slurry). Proteins were
immunoprecipitated with saturating amounts of Ab and 30 µL sepharose-coupled protein A (50% wt/vol slurry). Immunoprecipitated proteins were washed, subjected to sodium dodecyl sulfate (SDS) polyacrylamide electrophoresis, and immunoblotted as described under
Western blots.
Purification of nuclear and cytoplasmic cell fractions.
Cells (3 × 107) were treated as described above and
the pellet was lysed in 2 mL lysis buffer for 30 minutes at 4°C. The
nuclear and the cytoplasmic cell fraction were prepared exactly as
described in Busch.35
Western blots.
Supernatants from the various cell fractions described above were
electrophoresed on SDS-polyacrylamide gel and blotted on to a
Hybond-ECL nitrocellulose membrane (Amersham #RPN 2020D; Amersham,
Allerod, Denmark). The immunoblot was incubated with primary Ab
according to the manufactures instruction for 2 hours, followed by
incubation with 1/1,000 peroxidase-conjugated antimouse or antirabbit
Ig for 1 hour, washed, and developed by ECL (Amersham #RPN 2106) using
the manufacturer's instructions.
EMSA analysis.
Cells (107) were treated as described above and the cell
membrane was lysed in EMSA lysis buffer I for 30 minutes at 4°C. The nuclear fraction was pelleted and lysed in EMSA lysis buffer II for 30 minutes at 4°C. The protein concentration in the lysate was
determined using a bradford assay. The M67 hSIE oligo
(5 -TGCAGTCGACATTTCCCGTAAATCGTCGA-3, 3-CAGCTGTAAAGGGCATTTAGCAGCTACGT-5) was labeled with
 32P-ATP and incubated with 10 µg of the lysate for 15 minutes at 4°C. Protein/DNA complexes were separated on a
nondenaturing 6% polyacrylamide gel with 0.5 × TBE. Unlabeled
competitor DNA (50-fold excess) or blocking Ab (1 µL) was incubated
with the lysate 15 minutes before mixing with the labeled M67 probe.
Alternatively, proteins were precipitated from the nuclear extract
(EMSA lysis buffer II extract) with biotinylated M67 probe and 50 µL
avidin-coupled agarose. The precipitate was washed three times with PBS
and subjected to anti-Stat-3 Western blotting as described above.
Cell transfection.
Cells (3 × 106) were transiently transfected with a
Psg513 vector containing the sequence for Stat-3 or Stat-3. The
constructs were kindly provided by Dr E. Caldenhoven and are described
in detail previously.25,36 Transfections were made using 10 µg of DNA and 10 µL LipofectAMINE (Life Technologia, Roskilde,
Denmark), but otherwise in accordance with the manufacturer's
instructions. Cells were grown for 40 hours and subsequently assayed
for apoptosis and Stat-3 expression. Transfection efficiency was
measured by transfecting cells with a CD20 construct. The construct was
kindly provided by Dr K. Helin and Dr E. Harlow (The
Cancer Center of Massachusetts General Hospital, Boston, MA). CD20 is a
membrane protein that is exclusively expressed on B-cells and
follicular dendritic cells.37 Cells were labeled with
fluorescein isothiocyanate (FITC)-conjugated anti-CD20 antibody (DAKO;
F799) and the percentage of CD20+ cells was measured by
flow cytometry using a FACScan (Becton Dickinson,
Mountain View, CA).
Apoptosis analysis.
Apoptosis was in principle measured as described
previously.6,10 Cells (106) were stimulated
as described above. After 30 minutes of stimulation at 37°C, the
cells were resuspended in RPMI 1640 supplemented with 10%
heat-inactivated FCS (106 cells/mL) and cultured for 6 hours at 37°C. At the end of the culture period, the cells were
pelleted, washed once in 2 mL 0.03% saponin (Sigma #S7900) in PBS, and
reacted with 1 mL 0.4 µg/mL 7-aminoactinomycin D (7-AAD; Sigma
#A9400) in 0.03% saponin for 25 minutes at room temperature in the
dark. The samples were analyzed immediately by flow cytometry in a
FACScan (Becton Dickinson) using a logarithmic fluorescence scale. The
apoptosis data are presented as the percentage of sub-G1
staining where the relevant background staining is subtracted.
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RESULTS |
MHC-I ligation induces tyrosine phosphorylation of Tyk2, but not
Jak1-3.
In an attempt to define intracellular signal transduction pathways
responsible for alterations in the proliferation kinetics after
ligation of MHC-I, we focused on the involvement of the Jak/Stat
pathway, which recently was documented to influence cell cycle
kinetics.38
Lysates from MHC-I-ligated Jurkat T cells were immunoprecipitated with
specific Abs against the tyrosine kinases Tyk2, Jak1, Jak2, and Jak3.
The tyrosine phosphorylation of the different kinases was examined as a
marker for their activation. Figure 1 shows
that a small increment of Tyk2 phosphorylation was detected after 2 minutes of MHC-I cross-linking (Fig 1, lane 2), and maximal phosphorylation was seen after 5 minutes of stimulation (Fig 1, lane
3). In contrast, TCR/CD3 cross-linking did not induce significant tyrosine phosphorylation of the Tyk2 kinase (Fig 1, lane 5). The time-dependent phosphorylations of the Tyk2 kinase were not a result of
sequestering or degradation of the Tyk2, because similar amounts of
Tyk2 were immunoprecipitated (Fig 1, lower part). The inability of
TCR/CD3 to induce Tyk2 phosphorylation was not due to suboptimal
TCR/CD3 stimulation, because both MHC-I and TCR/CD3 antibodies were
titrated and used at saturating conditions (data not shown).
Furthermore, we have previously shown that the level of tyrosine
phosphorylated proteins is higher in Jurkat T cells after TCR/CD3
ligation compared with MHC-I ligation7 using antibody
concentrations similar to those in Fig 1. We were not able to observe
any detectable tyrosine phosphorylation of Jak1, Jak2, or Jak3 after
MHC-I ligation (data not shown).
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| Fig 1.
Immunoprecipitates of Tyk2 obtained from lysates of
Jurkat 76.25 cells after MHC-I or TCR/CD3 ligation. Cells
(3 × 107) were preincubated with PBS (lane 1) or
saturating amounts of anti-2M Ab (lanes 2 through 4)
before exposure to avidin for the indicated time (lanes 1 through 4) or
exposed to anti-TCR/CD3 Abs (lane 5). Precipitates were immunoblotted
with antiphosphotyrosine Ab (upper panel). Blots were stripped and
reprobed with anti-Tyk2 Ab (lower panel).
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MHC-I ligation induces tyrosine phosphorylation of Stat-3.
Because the activation of the Tyk2 kinase has been shown to be
implicated in the tyrosine phosphorylation of Stat proteins, we
investigated whether MHC-I cross-linking induced tyrosine
phosphorylation of Stat1-6. Figure 2 shows
immunoprecipitates of the 92-kD Stat-3 protein immunoblotted with
antiphosphotyrosine antibody; a strong tyrosine phosphorylation of
Stat-3 was observed after 5 minutes of MHC-I ligation (Fig 2, lane 2).
Despite a pronounced effect of TCR/CD3 ligation on the overall level of
tyrosine phosphorylation,7 no specific phosphorylation of
immunopurified Stat-3 was observed after TCR/CD3 ligation (Fig 2, lane
3). The results were not due to sequestering or degradation of the
Stat-3 protein, because similar amounts of Stat-3 were
immunoprecipitated in all experiments (Fig 2, lower part). Ligation of
MHC-I or TCR/CD3 did not induce detectable tyrosine phosphorylation of
Stat1, Stat2, Stat4, Stat5, or Stat6 (data not shown).
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| Fig 2.
Immunoprecipitates of Stat-3 obtained from lysates of
Jurkat 76.25 cells after MHC-I or TCR/CD3 ligation. Cells
(3 × 107) were preincubated with PBS (lane 1) or
saturating amounts of anti-2M Ab (lanes 2 and 4) before
exposure to avidin for 5 minutes (lanes 1, 2, and 4) and/or
exposed to anti-TCR/CD3 Abs for 5 minutes (lanes 3 and 4). Precipitates
were immunoblotted with antiphosphotyrosine Ab (upper panel). Blots
were stripped and reprobed with anti-Stat-3 Ab (lower panel).
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MHC-I ligation induces nuclear translocation of
tyrosine-phosphorylated Stat-3.
Tyrosine-phosphorylated Stat proteins are known to translocate to the
nucleus, where they function as DNA-binding transcription factors.22 To determine whether tyrosine-phosphorylated
proteins were translocated to the nucleus after MHC-I cross-linking, we prepared a highly purified nuclear fraction from Jurkat T cells. Figure
3 shows an antiphosphotyrosine immunoblot
of the nuclear fraction after MHC-I ligation. In contrast to the fast
induction of several tyrosine-phosphorylated proteins in whole cell
lysats after MHC-I ligation,7 only one single
phosphotyrosine protein at 92 kD was present in the nucleus (weakly
after 30 minutes of MHC-I ligation and more pronounced after 60 minutes
stimulation; Fig 3). This suggests that the 92-kD protein is tyrosine
phosphorylated in the cytoplasma and then subsequently
translocated to the nucleus.
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| Fig 3.
Phosphotyrosine blot of the nuclear fraction of Jurkat
76.25 cells after MHC-I ligation. Cells (3 × 107)
were preincubated with PBS (lane 1) or saturating amounts of anti-2M Ab (lanes 2 and 3) before exposure to avidin for
the indicated time. The separated proteins were immunoblotted with antiphosphotyrosine Ab.
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To examine whether the 92-kD nuclear translocated protein was identical
to the Stat-3 protein, a study with comparable kinetics, as described
above, was performed with anti-Stat-3 antibodies. The Stat-3 protein
was present at high concentrations in the cytoplasma (Fig
4A), regardless of cell activation.
Ligation of MHC-I molecules induced a substantial increase in the
nuclear Stat-3 concentration after 30 and 60 minutes of MHC-I ligation
(Fig 4B). The weak bands under Stat-3 are impurities of the nuclear
fraction that are not observed consistently. Similar studies were made
with Stat1, 2, 4, 5, and 6, but none of these proteins was detected in
the cell nucleus after MHC-I ligation (data not shown). From these
results we conclude that MHC-I ligation induces tyrosine
phosphorylation of cytoplasmic Stat-3 that subsequently translocated to
the cell nucleus.
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| Fig 4.
Anti-Stat-3 immunoblot of the cytoplasmic and nuclear
fraction of Jurkat 76.25 cells after MHC-I ligation. Cells
(3 × 107) were preincubated with PBS (lane 1) or
saturating amounts of anti-2M Ab (lanes 2 and 3) before
exposure to avidin for the indicated time. Cells were separated in a
cytoplasmic (A) or nuclear (B) fraction as described in the Materials
and Methods. The separated fractions were immunoblotted with
anti-Stat-3 Ab.
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MHC-I-induced Stat-3 binds to specific DNA sequences.
To examine whether the nuclear translocated tyrosine-phosphorylated
Stat-3 protein was able directly to associate with DNA, an EMSA
analysis was performed using the human serum-inducible element
(hSIE) probe M67. The hSIE DNA-probe has been shown to associate with several Stat proteins, including Stat-3.30
Figure 5A shows that nuclear proteins from
Jurkat T cells 30 minutes after ligation of their MHC-I molecules
contained proteins with affinity for the hSIE DNA probe (Fig 5A, lanes
3 and 4). The association was highly specific, because the association
with the labeled probe was completely inhibited by a 50-fold excess of
unlabeled probe (Fig 5A, lane 5). In contrast, ligation of the TCR/CD3
complex did not induce shifts in the mobility of the hSIE probe.
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| Fig 5.
(A) EMSA analysis of nuclear proteins bound to the M67
hSIE DNA probe after MHC-I or TCR/CD3 ligation. Cells
(107) were preincubated with PBS (lane 1) or saturating
amounts of anti-2M Ab (lanes 3 through 5) before
exposure to avidin for the indicated time (lanes 1 and 3 through 5) or
exposed to anti-TCR/CD3 Ab for 30 minutes (lane 2). Nuclear extracts
were subjected to EMSA analysis as described in the Materials and
Methods. The shifted DNA was blocked with 50-fold excess nonlabeled DNA
(lane 5). (B) The EMSA analysis was performed as described under (A),
but the nuclear extract was preincubated for 15 minutes with antibodies before the addition of the labeled M67 probe. Experiments shown in
lanes 1 and 2 were preincubated with anti-Stat-3 antibody and those
shown in lanes 3 and 4 with isotype control antibody (anti-ISGF-3). (C) Precipitation of Stat-3 from the nuclear extract. EMSA nuclear extract was made as described under (A) and precipitated with a
biotinylated M67 probe and avidin-coupled agarose as described in the
Materials and Methods. Cells (107) were preincubated with
PBS (lane 1) or saturating amounts of anti-2M Ab (lanes
2 through 4) before exposure to avidin for the indicated time (lanes 1 through 4). The precipitate were immunoblotted with anti-Stat-3 Ab.
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To verify that the shifted complex contained the Stat-3 molecule, an
inhibition experiment was performed. Figure 5B shows an EMSA analysis
in which the nuclear extract was treated with anti-Stat-3 or isotype
control antibodies before incubation with the labeled M67 probe. No
supershift was observed under these conditions, but the shifted band
disappeared after treatment with the anti-Stat-3 antibody (Fig 5B,
lane 2), implying that the anti-Stat-3 antibody and the M67 probe
compete for binding to the same or nearby epitopes on the Stat-3
protein. To show that Stat-3 directly associates with hSIE in nuclear
extracts from MHC-I ligated cells, Stat-3 was precipitated from the
nuclear extract with biotinylated M67 probe and avidin coupled agarose
and subsequently immunoblotted with anti-Stat-3 antibodies. Figure 5C
shows that the Stat-3 protein in nuclear extracts from MHC-I-ligated
cells interact with the M67 probe (Fig 5C, lanes 2 through 4). These
results show that the MHC-I-induced nuclear Stat-3 protein can
associate with the common Stat binding DNA sequence hSIE from the c-fos
promoter.
Overexpression of Stat-3 inhibits MHC-I-induced
apoptosis.
To assess the physiological function of Stat-3 activation after MHC-I
ligation, Jurkat T cells were transfected with a normal Stat-3
(Stat-3) or a Stat-3 construct. Stat-3 is an isoform that
lacks the transactivating domain, and overexpression dominantly inhibits Stat-3-induced transcriptional
activation.36 Apoptotic cells
have condensed DNA, which leads to a lower stainability with
7-AAD.39 Apoptotic cells are therefore routinely measured by flow cytometry as the percentage staining below the G1
DNA peak, where the relevant background staining is subtracted
(percentage of sub-G1 staining). The results in Fig 6 show
that transfection with Stat-3 significantly inhibits MHC-I-induced
apoptosis of Jurkat T cells, as opposed to cells transfected with
Stat-3 or a control CD20 construct. These results suggest that
Stat-3 activation after MHC-I ligation is involved in the subsequent
induction of apoptosis. Because Stat-3 is constitutively present in the
cells, it is not possible to measure the transient transfection
efficiency. To deduce the transfection efficiency, a CD20 construct was
transiently transfected in parallel into Jurkat T cells using the same
transfection procedures as with the Stat-3 constructs. The percentage
of CD20+ cells was measured by flow cytometry. Transfection
efficiency ranged from 35% to 45% (data not shown). MTT and
proliferation analyses showed no difference in the viability or
proliferation of Jurkat T cells transfected with either control or
Stat-3 or Stat-3 constructs (data not shown), implying that
Stat-3 transfection does not interfere with the endogenous
proliferation or viability of Jurkat T cells.
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| Fig 6.
Apoptosis measurement of MHC-I ligated Jurkat T cells.
Cells were either untransfected or transiently transfected with the empty vector, CD20, Stat-3, or Stat-3. Cells were transfected as
described in the Materials and Methods. Subsequently, apoptosis was
measured as described in the Materials and Methods. The results are
shown as the percentage of sub-G1 staining where the
relevant background values are subtracted.
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DISCUSSION |
MHC-I-induced signaling in T cells may lead to a number of functional
alterations, including apoptosis, alteration of cell proliferation,
cytokine secretion, and new surface phenotypes.2,5 To
further clarify distinct signal pathways operating after MHC-I ligation, the engagement of the Jak/Stat signal pathway in
MHC-I-induced signaling in human T cells was investigated in the
present work.
We provide here evidence that MHC-I ligation of human T cells induces
tyrosine phosphorylation of the Tyk2 tyrosine kinase. In contrast, none
of the other Jak family tyrosine kinases (Jak1, Jak2, or Jak3) was
activated after MHC-I ligation. Immunoprecipitation of the Stat-family
proteins Stat-1 through Stat-6 showed that the 92-kD Stat-3 protein was
tyrosine phosphorylated in response to MHC-I ligation, whereas no
detectable tyrosine phosphorylation of Stat1, Stat2, Stat4, Stat5, and
Stat6 was observed. In accordance with a previous study by Beadling et
al,40 we did not observe activation of proteins from the
Jak and Stat families after TCR/CD3 ligation.
Tyrosine-phosphorylated Stat proteins make homodimers and/or
heterodimers in the cytoplasm and are subsequently translocated to the
cell nucleus by a currently unknown mechanism.20,22 To
elucidate whether tyrosine-phosphorylated proteins were translocated to
the nucleus upon MHC-I ligation, a highly purified nuclear fraction was
obtained from Jurkat T-cell lysates. In the nuclear fraction, a 92-kD
tyrosine-phosphorylated protein was observed after 30 minutes of MHC-I
ligation that became more intensively phosphorylated after 60 minutes
of stimulation. Because the induction of tyrosine-phosphorylated
proteins in the cytolasma is observed within 5 seconds,7
this suggests that the protein was tyrosine phosphorylated in the
cytoplasma and subsequently translocated to the cell nucleus.
Studies were performed to elucidate whether the 92-kD protein was
identical to the Stat-3 protein. Immunoblotting of a purified nuclear
lysats with anti-Stat-3 antibodies showed that the Stat-3 protein was
indeed observed in the nucleus after 60 minutes of MHC-I ligation.
Together, these results strongly suggest that MHC-I ligation of T cells
induces tyrosine phosphorylation and subsequent nuclear translocation
of the Stat-3 transcription factor.
We tested whether nuclear supernatant from MHC-I-stimulated cells
could associate with the hSIE DNA probe derived from the c-fos
promoter, a DNA sequence known to associate with various activated Stat
proteins, including Stat-3.30 In a gel-shift assay (EMSA),
nuclear supernatant from MHC-I-ligated Jurkat T cells induced a clear
shift in the mobility of the hSIE probe, indicating an association
between the hSIE DNA probe and one or more of the proteins in the
nuclear supernatant. Furthermore, the shifted complex was absent in
nuclear extracts preincubated with anti-Stat-3 antibodies, and the
Stat-3 protein could be coprecipitated from nuclear cell extracts of
Jurkat T cells with a biotinylated hSIE probe and avidin-coupled
agarose. Together, these results demonstrate that the Stat-3 protein
from MHC-I-ligated cells specifically interacts with DNA representing
a common Stat binding sequence.
To investigate the functional consequences of MHC-I-induced Stat-3
activation, Jurkat T cells were transfected with constructs encoding
Stat-3 or Stat-3. When cells were transiently transfected with
the nontransactivating isoform Stat-3, a significant reduction in
the MHC-I-induced apoptosis of Jurkat T cells was observed. Because of
technical limitations, we were only able to transiently transfect about
40% of the cells; therefore, it is hard to judge whether the dominant
negatively acting Stat-3 totally abolish apoptosis after MHC-I
ligation or whether other signals participate. Nonetheless, the data
suggest that activated Stat-3 is involved at least in a significant
part of the intracellular signals leading to apoptosis after MHC-I
ligation. We tried to produce Jurkat T cells stabily transfected with
the Stat-3 construct, but, unfortunately, the attempts were not
successful.
Of particular interest, we have recently found that JNK activity is
critically involved in MHC-I-induced apoptosis.6 Thus, in
future studies, it will be of interest to elucidate the cooperation between JNK and Stat-3 in MHC-I-induced apoptosis.
It is well established that the cytoplasmic domain of the MHC-I
molecule is not essential for MHC-I-induced signal
transduction11,12; therefore, it is highly likely that the
MHC-I molecule associates with and uses the signal transduction devices
of other transmembrane molecules. The MHC-I molecules have been shown
to be either physically or functionally coupled to other signal
transducing proteins.2 This report presents evidence that
MHC-I ligation mediates signals through the Jak/Stat signal pathway. It
is therefore tempting to suggest that MHC-I molecules either directly
or indirectly associate with receptor(s) for growth regulatory
cytokine(s). An alternative explanation is that the MHC-I signal
induces secretion of intracellular stores of cytokines to the media,
which then mediate the activation of the TYK2 kinase and the Stat-3
protein through their respective receptors. However, this scenario is highly unlikely, because fresh Jurkat T cells exposed to
antibody-depleted culture supernatants from 2 to 10 minutes with
MHC-I-ligated cells did not show any tyrosine phosphorylation of
Stat-3 (data not included).
The MHC molecules associate both directly and functionally with the
IL-2 and IL-4 receptors16,17,41; therefore, these receptors
could be likely candidates through which the MHC-I molecule activates
TYK2 and Stat-3. To our knowledge, no single receptor has been shown to
exclusively phosphorylate TYK2 and Stat-3. This suggests that the MHC-I
molecules do not uncritically use cytokine receptors and mediate the
same signal as these; teleologically, this would also be very
inappropriate in a complex biological system. Rather, it is likely that
the MHC-I molecule by ligation induces specific changes in the receptor to which it associates and thereby induces a specific signal.
The precise physiological function of Stat proteins is currently not
well understood. One or more Stat proteins are activated in response to
almost all known cytokines. This suggests that Stat proteins are
involved in various physiological signals, as diverse as signals
leading to growth progression through cell cycle from G1 to
G2 to signals leading to growth inhibition and activation-induced cell death. Of particular interest, Toshio Hirano's
group has recently demonstrated that mutation of the Stat-3 binding
domain in the cytoplasmic part of the gp130 chain of the IL-6 receptor
abolished IL-6-induced growth arrest.42 Subsequently, the
same group showed that dominant-negative forms of Stat-3 inhibited
IL-6-induced growth arrest at
G0/G1.43 These observations are in
accordance with the observations on Jurkat T cells described in this
report and our recent data showing that MHC-I ligation induces a
particular anergic-like phenotype of the -chain and ZAP70 tyrosine
kinase,10 reduces colony formation in semisolid
culture,44 and is a key inducer of growth arrest and
apoptosis.4,6,10,45-47 We have previously shown that
staphylococcal enterotoxins exert a profound inhibitory effect on
IL-2-induced Jak3 and Stat protein activation,38 a process
dependent on MHC class II binding by the staphylococcal enterotoxin
(N.Ø., unpublished observation).
Even though there has been an enormous interest in the Jak/Stat signal
pathway, relatively little is known about its biological function. Our
hypothesis is that the MHC-I molecule uses other transmembrane
molecules for signal transduction, which may induce both
MHC-I-specific signals and modulation of cytokine receptor signaling.
The nature of this signal molecule(s) and the extent of its modulation
remain to be clarified.
| |
FOOTNOTES |
Submitted November 3, 1997;
accepted February 19, 1998.
Supported by the Danish Medical Research Council, the Novo Nordic
Foundation, the Beckett Foundation, the Lundbeck Foundation, the Leo
Research Foundation, and the Alfred Benzon Foundation. N.Ø. is
supported by the Danish Allergy Research Center.
Address reprint requests to Søren Skov, MSc, Cell
Cybernetics Laboratory, Institute of Medical Microbiology and
Immunology, The Panum Institute, University of Copenhagen, Blegdamsvej
3, Bldg 22-5, 2200 Copenhagen N, Denmark; e-mail:
s.skov{at}sb.immi.ku.dk.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Dr Erik Caldenhoven (University Hospital Utrecht,
Utrecht, The Netherlands) for providing the Stat-3 constructs and Tania
Palm and Mette Jeppesen for excellent technical assistance.
| |
REFERENCES |
1. Klein J: Natural History of the Major Histocompatibility Complex.
New York, NY, Wiley, 1986
2.
Tscherning T,
Claesson MH:
Signal transduction via MHC class-I molecules in T cells.
Scand J Immunol
39:117,
1994[Medline]
[Order article via Infotrieve]
3.
Takahashi H,
Nakagawa Y,
Leggatt GR,
Ishida Y,
Saito T,
Yokomuro K,
Berzofsky JA:
Inactivation of human immunodeficiency virus (HIV)-1 envelope-specific CD8 positive cytotoxic T lymphocytes by free antigenic peptide: A self-veto mechanism?
J Exp Med
183:879,
1996[Abstract/Free Full Text]
4.
Sambhara SR,
Miller RG:
Programmed cell death of T cells signaled by the T cell receptor and the alpha 3 domain of class I MHC.
Science
252:1424,
1991[Abstract/Free Full Text]
5.
Bregenholt S,
Röpke M,
Skov S,
Claesson MH:
Ligation of MHC class I molecules on peripheral blood T lymphocytes induces new phenotypes and functions.
J Immunol
157:993,
1996[Abstract]
6.
Skov S,
Klausen P,
Claesson MH:
Ligation of MHC class I molecules on human T-cells induces cell death through PI-3 kinase-induced c-Jun N-terminal kinase activity: A novel pathway distinct from Fas-induced apoptosis.
J Cell Biol
139:1523,
1997[Abstract/Free Full Text]
7.
Skov S,
Odum N,
Claesson MH:
MHC-class I signaling in T cells leads to tyrosine kinase activity and PLC gamma1 phosphorylation.
J Immunol
154:1167,
1995[Abstract]
8.
Dissing S,
Geisler C,
Rubin B,
Plesner T,
Claesson MH:
T cell activation. II. Activation of human T lymphoma cells by cross-linking of their MHC class I antigens.
Cell Immunol
126:196,
1990[Medline]
[Order article via Infotrieve]
9.
Geppert TD,
Wacholtz MC,
Patel SS,
Lightfoot E,
Lipsky PE:
Activation of human T cell clones and Jurkat cells by cross-linking class I MHC molecules.
J Immunol
142:3763,
1989[Abstract]
10.
Skov S,
Bregenholt S,
Claesson MH:
MHC class I ligation of human T-cells activates the ZAP70 and the P561ck tyrosine kinases, leads to an alternative phenotype of the TCR/CD3 zeta-chain, and induces apoptosis.
J Immunol
158:3189,
1997[Abstract]
11.
Gur H,
el-Zaatari F,
Geppert TD,
Wacholtz MC,
Taurog JD,
Lipsky PE:
Analysis of T cell signaling by class I MHC molecules: The cytoplasmic domain is not required for signal transduction.
J Exp Med
172:1267,
1990[Abstract/Free Full Text]
12. Gur H, Geppert TD, Lipsky PE: The cytoplasmatic domain is not
required for class I MHC molecules to aggregate, internalize, and
stimulate T cell activation. FASEB J 5:A990, 1991
13.
Due C,
Simonsen M,
Olsson L:
The major histocompatibility complex class I heavy chain as a structural subunit of the human cell membrane insulin receptor: Implications for the range of biological functions of histocompatibility antigens.
Proc Natl Acad Sci USA
83:6007,
1986[Abstract/Free Full Text]
14.
Centrella M,
McCarthy TL,
Canalis E:
Beta 2-microglobulin enhances insulin-like growth factor I receptor levels and synthesis in bone cell cultures.
J Biol Chem
264:18268,
1989[Abstract/Free Full Text]
15.
Schreiber AB,
Schlessinger J,
Edidin M:
Interaction between major histocompatibility complex antigens and epidermal growth factor receptors on human cells.
J Cell Biol
98:725,
1984[Abstract/Free Full Text]
16.
Sharon M,
Gnarra JR,
Baniyash M,
Leonard WJ:
Possible association between IL-2 receptors and class I HLA molecules on T cells.
J Immunol
141:3512,
1988[Abstract]
17.
Hansen NQ,
Tscherning T,
Claesson MH:
T-cell activation. IV. Evidence for a functional linkage between MHC class I, interleukin-2 receptor, and interleukin-4 receptor molecules.
Cytokine
3:35,
1991[Medline]
[Order article via Infotrieve]
18.
Firmbach-Kraft I,
Byers M,
Shows T,
Dalla-Favera R,
Krolewski JJ:
tyk2, prototype of a novel class of non-receptor tyrosine kinase genes.
Oncogene
5:1329,
1990[Medline]
[Order article via Infotrieve]
19.
Ihle JN,
Witthuhn BA,
Quelle FW,
Yamamoto K,
Silvennoinen O:
Signaling through the hematopoietic cytokine receptors.
Annu Rev Immunol
13:369,
1995[Medline]
[Order article via Infotrieve]
20.
Shuai K,
Horvath CM,
Huang LH,
Qureshi SA,
Cowburn D,
Darnell JE Jr:
Interferon activation of the transcription factor Stat91 involves dimerization through SH2-phosphotyrosyl peptide interactions.
Cell
76:821,
1994[Medline]
[Order article via Infotrieve]
21.
Darnell JE Jr,
Kerr IM,
Stark GR:
Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins [Review].
Science
264:1415,
1994[Abstract/Free Full Text]
22.
Ihle JN:
STATs: Signal transducers and activators of transcription.
Cell
84:331,
1996[Medline]
[Order article via Infotrieve]
23.
Nielsen M,
Svejgaard A,
Skov S,
Odum N:
Interleukin-2 induces tyrosine phosphorylation and nuclear translocation of stat3 in human T lymphocytes.
Eur J Immunol
12:3082,
1994
24.
Mu SX,
Xia M,
Elliott G,
Bogenberger J,
Swift S,
Bennett L,
Lappinga DL,
Hecht R,
Lee R,
Saris CJ:
Megakaryocyte growth and development factor and interleukin-3 induce patterns of protein-tyrosine phosphorylation that correlate with dominant differentiation over proliferation of mpl-transfected 32D cells.
Blood
86:4532,
1995[Abstract/Free Full Text]
25.
Caldenhoven E,
van Dijk T,
Raaijmakers JA,
Lammers JW,
Koenderman L,
De Groot RP:
Activation of the STAT3/acute phase response factor transcription factor by interleukin-5.
J Biol Chem
270:25778,
1995[Abstract/Free Full Text]
26.
Guschin D,
Rogers N,
Briscoe J,
Witthuhn B,
Watling D,
Horn F,
Pellegrini S,
Yasukawa K,
Heinrich P,
Stark GR,
Ihle JN,
Kerr IM:
A major role for the protein tyrosine kinase JAK1 in the JAK/STAT signal transduction pathway in response to interleukin-6.
EMBO J
14:1421,
1995[Medline]
[Order article via Infotrieve]
27.
Yin T,
Keller SR,
Quelle FW,
Witthuhn BA,
Tsang ML,
Lienhard GE,
Ihle JN,
Yang YC:
Interleukin-9 induces tyrosine phosphorylation of insulin receptor substrate-1 via JAK tyrosine kinases.
J Biol Chem
270:20497,
1995[Abstract/Free Full Text]
28.
Finbloom DS,
Winestock KD:
IL-10 induces the tyrosine phosphorylation of tyk2 and Jak1 and the differential assembly of STAT1 alpha and STAT3 complexes in human T cells and monocytes.
J Immunol
155:1079,
1995[Abstract]
29.
Novak U,
Harpur AG,
Paradiso L,
Kanagasundaram V,
Jaworowski A,
Wilks AF,
Hamilton JA:
Colony-stimulating factor 1-induced STAT1 and STAT3 activation is accompanied by phosphorylation of Tyk2 in macrophages and Tyk2 and JAK1 in fibroblasts.
Blood
86:2948,
1995[Abstract/Free Full Text]
30.
Zhong Z,
Wen Z,
Darnell JE Jr:
Stat3: A STAT family member activated by tyrosine phosphorylation in response to epidermal growth factor and interleukin-6.
Science
264:95,
1994[Abstract/Free Full Text]
31.
Leaman DW,
Pisharody S,
Flickinger TW,
Commane MA,
Schlessinger J,
Kerr IM,
Levy DE,
Stark GR:
Roles of JAKs in activation of STATs and stimulation of c-fos gene expression by epidermal growth factor.
Mol Cell Biol
16:369,
1996[Abstract/Free Full Text]
32.
Ezumi Y,
Takayama H,
Okuma M:
Thrombopoietin, c-Mpl ligand, induces tyrosine phosphorylation of Tyk2, JAK2, and STAT3, and enhances agonists-induced aggregation in platelets in vitro.
FEBS Lett
374:48,
1995[Medline]
[Order article via Infotrieve]
33.
Geisler C,
Plesner T,
Pallesen G,
Skjødt K,
Odum N,
Larsen JK:
Characterization and expression of the human T-cell receptor T3 complex by the monoclonal antibody F101.01.
Scand J Immunol
27:685,
1988[Medline]
[Order article via Infotrieve]
34.
Odum N,
Martin PJ,
Schieven GL,
Norris NA,
Grosmaire LS,
Hansen JA,
Ledbetter JA:
Signal transduction by HLA-DR is mediated by tyrosine kinase(s) and regulated by CD45 in activated T cells.
Hum Immunol
32:85,
1991[Medline]
[Order article via Infotrieve]
35.
Busch H:
Isolation and purification of nuclei.
Methods Enzymol
12:417,
1967
36.
Caldenhoven E,
van Dijk TB,
Solari R,
Armstrong J,
Raaijmakers JAM,
Lammers JWJ,
Koenderman L,
De Groot RP:
STAT3beta, a splice variant of transcription factor STAT3, is a dominant negative regulator of transcription.
J Biol Chem
271:13221,
1996[Abstract/Free Full Text]
37. Knapp W: Leucocyte Typing IV. White Cell Differentiation
Antigens. Oxford, UK, Oxford, 1989
38.
Nielsen M,
Svejgaard A,
Ropke C,
Nordahl M,
Odum N:
Staphylococcal enterotoxins modulate interleukin 2 receptor expression and ligand-induced tyrosine phosphorylation of the Janus protein-tyrosine kinase 3 (Jak3) and signal transducers and activators of transcription (Stat proteins).
Proc Natl Acad Sci USA
92:10995,
1995[Abstract/Free Full Text]
39.
Darzynkiewicz Z,
Bruno S,
Del Bino G,
Gorczyca W,
Hotz MA,
Lassota P,
Traganos F:
Features of apoptotic cells measured by flow cytometry [Review].
Cytometry
13:795,
1992[Medline]
[Order article via Infotrieve]
40.
Beadling C,
Guschin D,
Witthuhn BA,
Ziemiecki A,
Ihle JN,
Kerr IM,
Cantrell DA:
Activation of JAK kinases and STAT proteins by interleukin-2 and interferon alpha, but not the T cell antigen receptor, in human T lymphocytes.
EMBO J
13:5605,
1994[Medline]
[Order article via Infotrieve]
41.
Nielsen M,
Odum N,
Bendtzen K,
Ryder LP,
Jakobsen BK,
Svejgaard A:
MHC class II molecules regulate growth in human T cells.
Exp Clin Immunogen
11:23,
1994[Medline]
[Order article via Infotrieve]
42.
Yamanaka Y,
Nakajima K,
Fukada T,
Hibi M,
Hirano T:
Differentiation and growth arrest signals are generated through the cytoplasmic region of gp130 that is essential for Stat3 activation.
EMBO J
15:1557,
1996[Medline]
[Order article via Infotrieve]
43.
Nakajima K,
Yamanaka Y,
Nakae K,
Kojima H,
Ichiba M,
Kiuchi N,
Kitaoka T,
Fukada T,
Hibi M,
Hirano T:
A central role for Stat3 in IL-6-induced regulation of growth and differentiation in M1 leukemia cells.
EMBO J
15:3651,
1996[Medline]
[Order article via Infotrieve]
44.
Claesson MH,
Dissing S,
Tscherning T,
Geisler C:
T-cell activation. V. Anti-major histocompatibility complex class I antibody-induced activation and clonal abortion in Jurkat T-leukaemic cells.
Immunology
78:444,
1993[Medline]
[Order article via Infotrieve]
45.
Amirayan N,
Vernet C,
Machy P:
Class I-specific antibodies inhibit proliferation in primary but not secondary mouse T cell responses.
J Immunol
148:1971,
1992[Abstract]
46.
Smith DM,
Bluestone JA,
Jeyarajah DR,
Newberg MH,
Engelhard VH,
Thistlethwaite JR Jr,
Woodle ES:
Inhibition of T cell activation by a monoclonal antibody reactive against the alpha 3 domain of human MHC class I molecules.
J Immunol
153:1054,
1994[Abstract]
47.
Wallen-Ohman M,
Borrebaeck CA:
A cell surface antigen (BAL) defined by a mouse monoclonal antibody inducing apoptosis in a human lymphocytic leukemia cell line.
Int J Cancer
57:544,
1994[Medline]
[Order article via Infotrieve]
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Abstract
Introduction
Abstract