(Received for publication, December 28, 1995)
From the
The mechanism of action of the immunosuppressive drug
cyclosporin A (CsA) is the inactivation of the
Ca/calmodulin-dependent serine-threonine phosphatase
calcineurin by the drug-immunophilin complex. Inactive calcineurin is
unable to activate the nuclear factor of activated T cells (NFAT), a
transcription factor required for expression of the interleukin 2
(IL-2) gene. IL-2 production by CsA-treated cells is therefore
dramatically reduced. We demonstrate here, however, that NFAT can be
activated, and significant levels of IL-2 can be produced by the
CsA-resistant CD28-signaling pathway. In transient transfection assays,
both multicopy NFAT- and IL-2 promoter-
-galactosidase reporter
gene constructs could be activated by phorbol 12-myristate 13-acetate
(PMA)/
CD28 stimulation, and this activation was resistant to CsA.
Electrophoretic mobility shift assay showed the induction of a
CsA-resistant NFAT complex in the nuclear extracts of peripheral blood
T cells stimulated with PMA plus
CD28. Peripheral blood T cells
stimulated with PMA/
CD28 produced IL-2 in the presence of CsA.
Collectively, these data suggest that NFAT can be activated and IL-2
can be produced in a calcineurin independent manner.
Nuclear factor of activated T cells (NFAT) ()is
required for IL-2 production after antigenic stimulation of T
cells(1, 2) . Upon stimulation of T cells, an
NFAT-containing protein complex appears in the nucleus and recognizes
two or more sites in the IL-2 promoter(2, 3) . The
complex is composed of a pre-existing cytoplasmic component, which
translocates to the nucleus, and an inducible nuclear component
composed of members of the AP1
family(4, 5, 6) . Translocation of the
cytoplasmic component involves Ca
-induced activation
of the calcium/calmodulin-dependent serine/threonine phosphatase
calcineurin(5, 7) . Inactivation of calcineurin by the
immunosuppressive drugs CsA or FK506 results in the blockage of
translocation and inhibition of IL-2
production(7, 8) .
CD28 is a T cell surface
molecule that plays a major role as a coreceptor in the optimal
activation of T cells(9) . One of the major effects of CD28
stimulation is increased production of cytokines, such as IL-2,
interferon , granulocyte macrophage-colony stimulating factor, and
others, through transcriptional and post-transcriptional
mechanisms(10, 11) . One of the hallmarks of the CD28
costimulation is its insensitivity to the immunosuppressive drugs CsA
and FK506(10, 12, 13) . We reasoned that if
CD28 costimulation results in IL-2 production, and if NFAT is required
for the expression of IL-2, there must be a CsA-resistant pathway for
activation of NFAT. In this report, we demonstrate that NFAT can be
activated in a CsA-resistant manner.
Figure 1:
Activation
of an NFAT-reporter gene construct in the presence of CsA. A and B, the NFAT-TK-gal (10 µg) and TK-
gal
constructs (10 µg) were transiently transfected into Jurkat T cells
that were subsequently stimulated with medium alone, PMA,
PMA/
CD28, or PMA/ionomycin. Wherever indicated, cells were
pretreated with CsA (500 ng/ml) for 30 min. Results are the average of
two separate experiments. C, effect of overexpression of
NFAT
on NFAT-TK-
gal construct. NFAT-TK-
gal and
TK-
gal constructs (10 µg each) were transiently transfected
into Jurkat cells along with NFAT
expression vector (10
µg). The cells were subsequently stimulated as described above. The
average of two separate experiments is shown
here.
In the
experiment described above, it was possible that the assay measured
some other unknown factor able to bind to the NFAT sites in the
reporter plasmid rather than bona fide NFAT activity. To test
this, the assays were repeated in the presence of cotransfected
NFAT. Consistent with the results of
others(16, 21) , NFAT
had little effect on
the multicopy NFAT reporter in untreated cells (Fig. 1C), but had a marked effect in cells stimulated
with PMA and
CD28 (15-fold induction of NFAT activity relative to
the vector alone). As before, this induction was insensitive to CsA. In
the same assay, promoter activity induced by PMA + ionomycin was
abolished in the presence of CsA (data not shown). These results
indicate that overexpressed NFAT
protein can be activated
through a CsA-resistant pathway.
In order to determine whether the
IL-2 promoter can also be activated by a CsA-resistant pathway, the
568-base pair IL-2 promoter--galactosidase reporter construct
(designated as pIL2-568) was tested in the transient transfection
assay (Fig. 2). Like the multicopy NFAT-TK-
gal construct,
pIL2-568 showed much higher promoter activity after PMA/
CD28
stimulation compared to either the unstimulated control or to PMA alone (panel A). This activity was further increased in the presence
of overexpressed NFAT
protein (panel B). The
vector alone did not have any activity (data not shown). The inducible
promoter activity was completely resistant to CsA (panels A and B), whereas CsA suppressed PMA/ionomycin-inducible
promoter activity (panel C). To test whether the promoter
activity induced by PMA +
CD28 was mediated by NFAT, a
dominant-negative NFAT
protein (16) was tested in
transient transfection assay. As shown in Fig. 2, A and B, overexpression of the dominant negative resulted in
70-80% inhibition of PMA/CD28-inducible promoter activity, but,
as expected, the dominant negative NFAT
protein had very
little effect on an NF
B-TK-
gal reporter construct (panel
D). These data demonstrate that the IL-2 promoter can be activated
by PMA/
CD28 in a CsA-resistant manner, and that this activation is
mediated by NFAT.
Figure 2:
Activation of an IL-2
promoter--galactosidase reporter gene construct in the presence of
CsA. A, the pIL2-568 reporter gene (5 µg) along with
expression vector alone or in combination with the expression vector
for a dominant negative form of NFAT
(5 µg) was
transiently transfected into Jurkat cells and was subsequently
stimulated as described above. B, the pIL2-568 reporter
gene along with the NFAT
expression vector (5 µg each)
in the presence or absence of the dominant negative NFAT
(5
µg) was transfected into Jurkat cells that were subsequently
stimulated as described in Fig. 1. The amount of transfected
plasmids was normalized by adding the empty vector, pBJ5. C,
similar experiment as B except the stimulation was with PMA
and ionomycin. D, the NF
B-TK-
gal reporter gene was
transfected into Jurkat cells along with the expression vector for the
dominant negative NFAT
(5 µg each). Cells were
subsequently stimulated as described in Fig. 1.
Figure 3:
EMSA demonstrating the presence of
inducible NFAT complex. A, EMSA with P-labeled
IL-2 distal NFAT site using nuclear extracts of Jurkat cells treated
with medium (lane 1), PMA/CD28 (lane 2), PMA/CD28/CsA (lane 3), PMA/ionomycin (PMA/Iono.; lane 4),
and PMA/ionomycin/CsA (lane 5). Lanes 6, 7,
and 8 represent competition analysis of PMA/CD28-treated
nuclear extracts using unlabeled specific competitor (S.C.),
AP1 oligonucleotide, and SP1 oligonucleotide as a nonspecific
competitor (N.C.). B, supershift analysis of
PMA/CD28-inducible complex (lane 1) using
NFAT (lane
2),
NFAT
(lane 4), and an irrelevant
antiserum (IR, lane 3). C, EMSA with labeled
IL-2 NFAT site using nuclear extracts of peripheral blood T cells
treated with medium alone (lane 1), PMA/CD28 (lane
2), PMA/CD28/CsA (lane 3), PMA/ionomycin (lane
10), and PMA/ionomycin/CsA (lane 11). Lanes 4, 5, and 6 represent competition analysis of
PMA/CD28-treated nuclear extracts using unlabeled specific competitor (S.C.), AP1 site, and SP1 as a nonspecific competitor (N.C.), respectively. Lanes 7, 8, and 9 represent supershift analysis of PMA/CD28-inducible complex using
NFAT,
NFAT
, and an irrelevant antiserum (IR), respectively.
Figure 4: Production of IL-2 by peripheral blood T cells. Peripheral blood T cells isolated from two different donors were stimulated with medium alone, PMA/CD28, PMA/CD28/CsA, PMA/ionomycin, and PMA/ionomycin/CsA for 24 h. IL-2 secreted into the medium was measured by enzyme-linked immunosorbent assay. CsA was added to the cells 30 min prior to stimulation.
Previous studies have shown that the immunosuppressive drug
CsA drastically inhibits, but does not completely block, IL-2
production following stimulation by CD3 and
CD28(10) . The drug-resistant activity can most likely be
traced to the CD28 signaling pathway, since stimulation of cells with
PMA (to mimic the Ca
-independent aspects of
CD3
signaling) and
CD28 is entirely resistant to CsA(10) . In
this case, IL-2 is produced at significant levels in the continued
presence of CsA or FK506. Since the transcription factor NFAT is
required for expression of the IL-2 gene, these results imply that, in
addition to the well-known Ca
-dependent pathway, NFAT
can be activated by a CsA-resistant pathway. Data presented here show
that this is the case.
The involvement of NFAT in the activation of
reporter gene constructs (both NFAT-TK-gal and pIL2-568)
after PMA/
CD28 treatment in the presence of CsA was demonstrated
by the facts that (i) overexpressed wild type NFAT augmented reporter
activity only after PMA/
CD28 stimulation, and (ii) the inducible
reporter activity was essentially eliminated in the presence of a
dominant negative mutant of NFAT. The presence of NFAT in the
PMA/
CD28-inducible DNA-protein complexes was also demonstrated by
the supershift assays. In agreement with the transient transfection
assays, the peripheral blood T cells were also shown to produce IL-2
after PMA/
CD28 treatment, and this production was resistant to
CsA.
The established pathway for NFAT activation involves
Ca-dependent activation of the serine-threonine
phosphatase calcineurin. Subsequent dephosphorylation of NFAT leads to
its nuclear translocation and, in conjunction with AP-1, DNA binding.
The data presented here suggest an alternate pathway for NFAT
activation that is calcineurin independent. Since dephosphorylation of
NFAT appears to be necessary for its activity, a different phosphatase
must be involved in this pathway. A candidate second messenger (instead
of Ca
) is ceramide, which has been reported to be
involved in CD28 signaling(22, 23) . Interestingly, a
serine/threonine phosphatase has been reported recently that is
involved in the sphingomyelin pathway(24) . It is an attractive
hypothesis that this or another ceramide-induced phosphatase can lead
to NFAT activation in a CsA-resistant manner. This idea is currently
being tested.
The present finding may explain the ineffectiveness of CsA in the treatment of graft versus host disease following allogenic bone marrow transplantation(25, 26) . The cytotoxic T cells thought to be responsible for graft versus host disease (27) express CD28 on their surface and are capable of producing cytokines responsible for immune reaction in the presence of CsA.