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Lymphocyte Cell Biology Section, Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal Diseases, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production by T cells and NK cells; however, the mechanisms of
inhibition have not been clearly defined. It has been suggested by some
studies that TGF-ß blocks cytokine-induced Janus kinase (JAK) and
STAT activation, as in the case of IL-2. In contrast, other studies
with cytokines like IFN- have not found such an inhibition. The
effect of TGF-ß on the IL-12-signaling pathway has not been
addressed. We examined this and found that TGF-ß1 did not have any
effect on IL-12-induced phosphorylation of JAK2, TYK2, and STAT4
although TGF-ß1 inhibited IL-2- and IL-12-induced IFN- production.
Similarly, but in contrast to previous reports, we found that TGF-ß1
did not inhibit IL-2-induced phosphorylation of JAK1, JAK3, and STAT5A.
Furthermore, gel shift analysis showed that TGF-ß1 did not prevent
activated STAT4 and STAT5A from binding to DNA. Our results demonstrate
that the inhibitory effects of TGF-ß on IL-2- and IL-12-induced
biological activities are not attributable to inhibition of activation
of JAKs and STATs. Rather, our data suggest the existence of
alternative mechanisms of inhibition by TGF-ß. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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secretion and enhances the
cytolytic activity of NK cells and cytotoxic T cells. Importantly, it
also drives the differentiation of naive CD4+ cells to Th1
cells 1, 2 . The role of IL-12 in cell-mediated immunity is
exemplified by IL-12 knockout mice, which are impaired in their ability
to generate a normal Th1 response and to produce IFN- 3 .
IL-12 transmits signals by binding to a receptor complex composed of
two subunits, ß1 and ß2 4 . Both subunits are required for the
formation of high affinity receptors and subsequent signaling 4, 5 .
Like other type I cytokine receptors, IL-12 receptor subunits do not
possess intrinsic tyrosine kinase activity but rather function by
associating with the members of the Janus family of kinases
(JAK)2, 6 . IL-12 induces
tyrosine phosphorylation of JAK2 and TYK2 7 , which in turn
phosphorylate other substrates including the transcription factors
STAT4 and STAT3 8, 9 . Activated STATs translocate to the nucleus and
regulate gene transcription 6, 10 . Whether STATs are directly
involved in exerting the biological effects of IL-12, such as IFN-
production, is presently not clear, and our knowledge of IL-12 target
genes is far from complete.
While cytokines like IL-12 and IL-2 promote immune responses, other
cytokines like TGF-ß suppress immune cell function. TGF-ß1 inhibits
IL-2- and IL-12-induced effects, including proliferation, IFN-
production, and the cytotoxic activity of NK cells and T cells
11, 12, 13, 14, 15, 16, 17 . Deficiency of TGF-ß1 results in severe immune
dysregulation, as evidenced by TGF-ß1 knockout mice, which develop
severe inflammatory disease in several organs and die of overt
autoimmune reaction 18, 19 .
An issue of great importance is how the immunosuppressive effects of
TGF-ß are mediated. Although there is ample documentation of the
inhibitory effects of TGF-ß, an understanding of the mechanism by
which these effects on immune cell function occur is lacking.
Conceivably, one mechanism might be that TGF-ß interferes with early
signaling events induced by cytokines; specifically, it could be
envisioned that TGF-ß might inhibit JAK or STAT activation. However,
several studies on the effects of TGF-ß on activation of JAKs and
STATs have provided conflicting results. TGF-ß1 was shown to inhibit
IL-5-induced activation of JAK2 and STAT1, and this was suggested to
underlie the inhibitory action of TGF-ß on eosinophils 20 .
Similarly, TGF-ß was reported to inhibit IL-2-induced activation of
JAKs and STATs 21, 22 , although the exact findings of these
two studies were somewhat contradictory. Han et al. 22 reported
inhibition of IL-2-induced phosphorylation of JAK1, JAK3, STAT5, and
STAT3 by TGF-ß1. However, Bright et al. 21 observed inhibition of
only JAK1 and STAT5 and no inhibition of IL-2-induced phosphorylation
of JAK3 or STAT3. In contrast, the suppression of IFN--induced class
II MHC gene expression by TGF-ß2 was found not to be the result
of inhibition of phosphorylation of JAKs or STATs 23 , but rather due
to inhibition of class II trans-activator (CIITA) 24, 25 .
The effect of TGF-ß on IL-12 signaling has not been previously reported. In light of the conflicting reports in different systems, it was of interest to address this issue. We found that TGF-ß1 had no effect on IL-12-induced phosphorylation of JAK2, TYK2, and STAT4. We also found that TGF-ß1 did not inhibit activated STAT4 from binding DNA. Thus, in contrast to what has been reported previously for some cytokines, the inhibitory effect of TGF-ß on IL-12-induced activities is not explained by an effect on activation of JAKs and STATs. More surprisingly, in contrast to previous reports, we found that TGF-ß1 did not inhibit IL-2-induced activation of JAKs and STATs in human T cells and an NK cell line. These studies suggest that alternative mechanisms, beyond inhibition of proximal signaling elements, are likely to account for the critical immunologic functions of TGF-ß.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant human TGF-ß1 and IL-12, and human IFN-
immunoassay kits were purchased from R&D Systems (Minneapolis, MN).
Human IL-2 was provided by Dr. C. Reynolds (National Cancer Institute,
Frederick, MD). Monoclonal anti-phosphotyrosine Ab (clone 4G10) and
polyclonal anti-JAK2 and -TYK2 Abs were purchased from Upstate
Biotechnology (Lake Placid, NY). Monoclonal anti-TYK2 Ab was
purchased from Transduction Laboratories (Lexington, KY). Polyclonal
rabbit anti-STAT4 Ab was purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). Rabbit polyclonal anti-JAK1 Ab was provided by
Dr. A. Larner (Cleveland Clinic Foundation, Cleveland, OH). The rabbit
polyclonal Abs against human JAK3 and STAT5A were raised in our
laboratory 26, 27 . RNA extraction kit (RNAgents Total RNA Isolation
System, Promega, Madison, WI) and RiboQuant Multiprobe RNase Protection
System (PharMingen, San Diego, CA) were purchased.
Cell culture
The IL-12-responsive human NK cell line NK3.3 was provided by
Dr. J. Kornbluth (Arkansas Cancer Research Center, Little Rock, AR) and
was cultured as described previously 7, 8 . PBMC were isolated from
peripheral blood of normal healthy donors by Ficoll-Paque gradient
centrifugation, activated with PHA (2 µg/ml) for 72 h, and
cultured for an additional day in presence of IL-2 (40 IU/ml).
Typically, this resulted in 
95% CD3+ cells. Before
stimulation with cytokine, cells were washed with acidified medium (pH
6.4) and rested overnight in RPMI 1640 containing 1% FCS.
Preparation of cytoplasmic and nuclear extracts
Cytoplasmic and nuclear extracts were prepared as previously described 28 with some modifications. Cytokine-stimulated cells were washed in 1x PBS containing 1 mM EDTA and 2 mM Na3VO4 and incubated at 4°C for 5–10 min in 1 ml of hypotonic solution (20 mM HEPES (pH 7.9), 10 mM KCl, 1 mM EDTA, 1.5 mM MgCl2, 10% glycerol, 0.2% Nonidet P-40, 2 mM Na3VO4, 0.5 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM DTT). Cells were homogenized with 16 strokes in a Dounce homogenizer. The homogenates were transferred to a microfuge tube and centrifuged at 1500 rpm for 5 min. Supernatants were saved and used as cytoplasmic extract. The crude nuclei were washed twice with 200 µl of hypotonic solution and centrifuged at 1500 rpm for 3 min. The nuclei were then resuspended in high salt buffer (20 mM HEPES (pH 7.9), 420 mM NaCl, 1 mM EDTA, 1.5 mM MgCl2, 20% glycerol, 2 mM Na3VO4, 0.5 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM DTT) and incubated at 4°C for 30 min, followed by incubation at room temperature for 10 min. Two hundred units of DNase I (Boehringer Mannheim, Indianapolis, IN) was added and incubated at room temperature for 10 min. The samples were centrifuged at 14,000 rpm at 4°C for 5 min, and supernatants were collected as nuclear extracts.
Immunoprecipitation and immunoblotting
Rested cells (3 x 107 NK3.3 cells or 5 x 107 T cells) were incubated at 37°C with TGF-ß1 (10 ng/ml) in 12 ml media for various times, followed by stimulation with IL-2 or IL-12 for 15 min at 37°C. Stimulated cells were washed and lysed with buffer containing 0.5% Triton X-100 29 . Clarified lysates were immunoprecipitated with either anti-STAT4, -STAT5A, -JAK1, -JAK2, -TYK2, or -JAK3 antisera conjugated to protein A-coupled Sepharose beads. The immunoprecipitates were resolved on 8% SDS-polyacrylamide gel and transferred to Immobilon (Millipore, Bedford, MA).
Immunoblotting with antiphosphotyrosine was conducted after blocking the membrane in Tris-buffered saline containing 1% fish gelatin, 2% goat serum, 0.1% BSA, and 0.5% Tween by sequential incubation with anti-phosphotyrosine Ab, biotinylated goat anti-mouse IgG, and HRP-conjugated streptavidin (Oncogene Science, Cambridge, MA). The phosphoproteins were detected by use of enhanced chemiluminescence (ECL, Amersham, Arlington Heights, IL).
Immunoblotting with Abs to the JAKs or STATs was conducted by blocking the membrane in TBS containing 5% nonfat dried milk and 0.1% Tween and incubating sequentially with primary Ab, HRP-conjugated goat anti-rabbit IgG (Boehringer Mannheim) or HRP-conjugated sheep anti-mouse IgG and detected by use of ECL. Before reprobing, the membranes were stripped in 62.5 mM Tris-HCl (pH 6.8) containing 2% SDS and 0.7% 2-ME at 70°C.
EMSA
Cell extracts were prepared from cytokine-stimulated NK3.3
cells, and EMSA was performed as described 30, 31 , using a
32P-labeled double-stranded oligonucleotide corresponding
to the GRR of the human FcRI
(5'-AGCATGTTTCAAGGATTTGAGATGTATTTCCCAGAAAAG-3') for IL-12 studies and
the GAS-like element of the CD23 promoter
(5'-AAGACCATTTCTAAGAAATCTATC-3') for IL-2 studies. Cell extracts were
incubated with the labeled oligonucleotide for 20 min at room
temperature. In supershift assays, this complex was further incubated
with anti-STAT4 Ab (5 µl) at 4°C for 30 min. The complexes were
electrophoresed through a 4.5% nondenaturing acrylamide gel and
subjected to autoradiography.
RNase protection assay
Total RNA was extracted from NK3.3 cells after preincubation of
cells with TGF-ß1 (10 ng/ml) for 15 min and stimulated with IL-2 or
IL-12 for 4 h. RNase protection assay was conducted as follows.
32P-labeled RNA probe was synthesized by incubating probes
from the multiprobe template set with T7 RNA polymerase for 1 h.
DNA was digested with 2 units of DNase I (Boehringer Mannheim), and RNA
probes were extracted with phenol and chloroform and precipitated with
ethanol. Labeled RNA probes were hybridized overnight at 56°C with
equal amounts of target RNA (5 µg), following which free probe and
single-stranded RNA were digested with T1 RNase (Life Technologies,
Gaithersburg, MD). The protected mRNA fragment was extracted with
phenol and chloroform, precipitated with ethanol, resolved on a 6%
denaturing polyacrylamide gel, and subjected to autoradiography.
IFN- transcript was identified by the length of the protected
fragment. Equivalent RNA loading was ascertained by using probes
derived from two housekeeping genes, namely, L32 and GAPDH.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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induced by IL-12 and
IL-2
The inhibitory effect of TGF-ß1 on T cell proliferation and
production of IFN- by IL-12 and IL-2 has been reported previously
11, 13, 15, 17, 32 . To establish that our system responded
appropriately to TGF-ß1 and to verify the activity of TGF-ß1 used,
we treated NK3.3 cells with TGF-ß1 and stimulated with varying
concentrations of IL-12 or IL-2 for 24 h and assayed cell-free
supernatants for IFN-. Consistent with previous findings, both IL-12
and IL-2 induced IFN- production in NK3.3 cells (Fig. 1
, A and B). IL-12
stimulation resulted in modest induction of IFN- (Fig. 1, A and B) whereas IL-2 was a more potent inducer
of IFN- production (Fig. 1B). When a combination of both
IL-2 and IL-12 was used, a synergistic effect on IFN- production was
observed (Fig. 1B). Treatment with TGF-ß1 resulted in
approximately 30–45% inhibition of IFN- induction by either IL-12
(Fig. 1, A and B) or IL-2 (Fig. 1B) or
the combination of both (Fig. 1B). To confirm that similar
results would be obtained with primary human cells, we determined the
effect of TGF-ß1 on activated T lymphocytes since activated, but not
resting, T cells express IL-12 receptors 33 . We found that both IL-2
and IL-12 induced IFN- production in activated T cells and that the
combination of IL-2 and IL-12 was synergistic (Fig. 1C).
Treatment with TGF-ß1 resulted in partial inhibition of IFN-
production by IL-2 or IL-12 separately or in combination (Fig. 1C).
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mRNA. To this end, we conducted RNase protection assay. As
shown in Fig. 1
D, stimulation with IL-2 or IL-12
(lanes 3 and 5, respectively)
resulted in increased amounts of IFN- transcript as compared with
control (lane 1). In accordance with the data
shown in Fig. 1, A-C, IL-2 was a more potent
inducer of IFN- transcripts than IL-12, and the combination of IL-12
and IL-2 showed a synergistic effect. Treatment with TGF-ß1 resulted
in a decrease in the induction of IFN- mRNA by IL-2 and IL-12
separately (lanes 4 and 6,
respectively) or in combination (lane 8). Equal
amounts of GAPDH and L32 transcripts confirmed that equal amounts of
target RNA was used. Thus, TGF-ß1 inhibits steady state levels of
IFN- message. To confirm that the TGF-ß1 used in our experiments
was active, we also conducted a standard growth inhibition assay of
CCL64 mink lung epithelial cells and found that TGF-ß1 inhibited
maximal growth at concentration below 0.5 ng/ml (data not shown). TGF-ß1 does not inhibit IL-12-induced phosphorylation of JAK2 and TYK2
One mechanism by which TGF-ß1 could inhibit the function of
IL-12 is by inhibition of the early signaling steps regulated by IL-12.
The most proximal event thus far defined in IL-12 signaling is the
phosphorylation of JAK2 and TYK2 7 . Therefore, we first looked to see
whether the phosphorylation of these kinases was inhibited by TGF-ß1.
NK3.3 cells were treated with TGF-ß1 for various times and stimulated
with IL-12. Cell lysates were immunoprecipitated with Abs to JAK2 or
TYK2, and the immunoprecipitates were blotted with
anti-phosphotyrosine. As seen in Fig. 2, stimulation of cells with IL-12
resulted in phosphorylation of JAK2 (lane 2),
while treatment with TGF-ß1 alone did not result in phosphorylation
of JAK2 (lane 8). Preincubation of cells with
TGF-ß1 for 10 min to 18 h had no effect on phosphorylation of
JAK2 (Fig. 2A, lanes 3-7).
Similarly, IL-12 induced phosphorylation of TYK2 (Fig. 2B,
lane 2), and treatment of cells with TGF-ß1 had no
effect on IL-12-induced phosphorylation of TYK2 (Fig. 2B,
lanes 3-7). To confirm that equal amounts
of JAK2 or TYK2 were loaded, the above blots were stripped and
reblotted with Abs to JAK2 or TYK2 (Fig. 2, A and
B, lower panels). To be certain that this
was also true in primary human cells, we conducted the same experiment
in activated T cells and found no inhibition of IL-12-induced
phosphorylation of TYK2 (Fig. 2C) or JAK2 (data not shown)
by TGF-ß1. These data demonstrate that the proximal event in
IL-12-induced JAK-STAT pathway, namely, phosphorylation of JAK2 and
TYK2, was unaffected by TGF-ß1; furthermore, the expression of these
proteins was neither inhibited nor enhanced by TGF-ß1.
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Activated JAKs phosphorylate cytokine receptors, thus providing
docking sites for STATs 34, 35 . STATs are also phosphorylated by
JAKs, allowing them to dimerize, translocate to the nucleus, and
activate gene transcription 6, 10 . Since the initial events in IL-12
signaling were not inhibited by TGF-ß1, we looked to see whether a
subsequent downstream signaling event, namely, the phosphorylation of
STAT4, was inhibited by TGF-ß1. As shown in Fig. 3, stimulation of cells with IL-12
resulted in phosphorylation of STAT4 (lane 2),
and, as expected, treatment with TGF-ß1 alone did not result in
phosphorylation of STAT4 (lane 8). Incubation
of cells with TGF-ß1 for 10 min to 18 h followed by stimulation
with IL-12 did not inhibit phosphorylation of STAT4 (Fig. 3A, lanes 3-7) as compared with
control cells (Fig. 3A, lane 2). That the
levels of STAT4 in these immunoprecipitates were equal was determined
by stripping the blot and reblotting with anti-STAT4 Ab (Fig. 3A, lower panel). Thus, treatment with
TGF-ß1 affected neither the level of expression of STAT4 nor its
phosphorylation. As before, we also conducted the same experiment with
activated human T cells and found that TGF-ß1 did not inhibit
IL-12-induced phosphorylation of STAT4 (Fig. 3B,
lanes 3-7).
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C,
IL-12-induced phosphorylation of STAT4 (Fig. 3C, lanes
5, 9, and 13) and treatment with
various concentrations of TGF-ß1 had no effect on IL-12-induced
phosphorylation of STAT4, regardless of the amount of IL-12 (Fig. 3C, lanes 6-8,
10-12, and 14-16). We
further tested a potential effect of TGF-ß1 on a broad time course of
STAT4 activation. NK3.3 cells were treated with TGF-ß1 for 30 min
followed by stimulation with IL-12 for various times (15 min-22 h). As
shown in Fig. 3D, STAT4 is rapidly phosphorylated by IL-12,
and this persists up to 22 h (lanes 3,
5, 7, 9, 11, and
13); treatment with TGF-ß1 (lanes
4, 6, 8, 10, and
14) did not inhibit IL-12-induced phosphorylation of STAT4.
Thus TGF-ß1 did not inhibit IL-12-induced STAT4 phosphorylation at
any dose that we tested nor did it alter the kinetics of STAT4
activation.
We have previously shown that STAT4 undergoes serine phosphorylation in
response to stimulation with IL-12; this is manifested by a retardation
in mobility and may be important in regulating transcriptional
activation 36 . Therefore, we considered the possibility that TGF-ß1
might inhibit serine phosphorylation of STAT4, even though tyrosine
phosphorylation of STAT4 was not inhibited by TGF-ß1. As seen in Fig. 3, A and B (lanes
3-7), and Fig. 3C (lanes
6-8, 10-12, and
14-16) the appearance of the slower migrating
form of STAT4 was not affected by TGF-ß1 in NK3.3 cells and primary T
cells. Furthermore, TGF-ß1 failed to inhibit IL-12-dependent
trans-activation of a STAT reporter construct (data not
shown), an event that is thought to be dependent upon tyrosine and
serine phosphorylation 36 . These data argue against TGF-ß1
inhibition of serine phosphorylation of STAT4. However, in view of the
fact that sites of serine phosphorylation on STAT4 have not been
mapped, we cannot exclude with certainty that TGF-ß1 might affect
this modification.
Activation of STATs by tyrosine phosphorylation is necessary for
dimerization and binding to DNA 37, 38 . Since IL-12-induced
phosphorylation of STAT4 was not inhibited by TGF-ß1, we expected
that TGF-ß1 would have no effect on IL-12-induced DNA binding
complex. As further proof of the lack of effect of TGF-ß1 on
IL-12-mediated STAT activation, we conducted an EMSA. Extracts from
cells stimulated with IL-12 showed binding to a 32P-labeled
double-stranded oligonucleotide corresponding to the GRR of the FcRI
(Fig. 4A, lane
3) while unstimulated cells did not (Fig. 4A,
lane 1). Supershift of the GRR binding complex on
addition of antiserum to STAT4 showed that the DNA binding complex
contained STAT4 (Fig. 4A, lanes 4,
6, 8, 10, and 12).
Treatment with TGF-ß1 for varying times before stimulation with IL-12
failed to inhibit the formation of STAT4 GRR complex (Fig. 4A, lanes 5, 7, 9,
and 11). We also considered the possibility that TGF-ß1
might inhibit nuclear targeting of STAT4. To investigate this, NK3.3
cells were pretreated with TGF-ß1 and stimulated with IL-12 (Fig. 4, B and C). STAT4 was immunoprecipitated from
cytoplasmic and nuclear extracts, and the immunoprecipitates were
blotted with anti-phosphotryrosine. As seen in Fig. 4, B
and C, unstimulated cells showed minimal phosphorylation of
STAT4 in both cytoplasmic and nuclear extracts. Importantly, most of
STAT4 protein was present in the cytoplasmic extract of unstimulated
cells (Fig. 4, B and C, lane 1),
with negligible amounts in the nuclear extract (Fig. 4, B
and C, lane 5) whereas, upon stimulation,
STAT4 was readily detected in the nucleus (Fig. 4B,
lane 6, and 4C, lane 7).
On stimulation with IL-12, phospho-STAT4 was detected in both cytoplasm
and nucleus. As seen in Fig. 4, B and C,
pretreatment with TGF-ß1 did not prevent nuclear
translocation or phosphorylation of STAT4 at times ranging from 15
min to 24 h. Taken together, the data presented in Figs. 2, 3, and 4 clearly argue that TGF-ß1 does not directly inhibit IL-12-induced
activation of JAKs and STATs.
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Since previous reports had suggested that IL-2-induced
phosphorylation of JAKs and STATs could be inhibited by TGF-ß1 21, 22 , we sought to determine whether IL-12 and IL-2 signaling differed
in this respect. We therefore investigated the effect of TGF-ß1 on
the IL-2-induced phosphorylation of JAK3, JAK1, or STAT5A. As shown in
Fig. 5, A and B,
IL-2 induced phosphorylation of JAK3 and JAK1 (Fig. 5, A and
B, respectively, lane 2). Treatment with
TGF-ß1 for varying times, however, did not result in inhibition of
IL-2-induced phosphorylation of either JAK3 or JAK1 (Fig. 5, A and B, respectively, lanes
3-7). Similarly, IL-2 induced phosphorylation of
STAT5A (Fig. 5C, lane 2). Treatment with
TGF-ß1 did not result in inhibition of IL-2-induced phosphorylation
of STAT5A (Fig. 5C, lanes 3-6).
Since these results in NK cells contradicted previous reports 21, 22 ,
we sought to confirm the result in primary human T cells. As shown in
Fig. 5, IL-2 induced phosphorylation of JAK3 and JAK1
(lane 2 in Fig. 5, D and
E, respectively). Treatment with TGF-ß1 did not result in
inhibition of IL-2-induced phosphorylation of either JAK3 or JAK1 (Fig. 5, D and E, respectively, lanes
3-7) in T cells. Further, TGF-ß1 did not affect
IL-2-induced phosphorylation of STAT5A in primary T cells (Fig. 5F, lanes 3-7).
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, lane
2), while unstimulated cells did not (Fig. 6, lane
1). Treatment with TGF-ß1 for the indicated
times before stimulation with IL-2 did not inhibit formation of a
GAS binding complex (Fig. 6, lanes 3-8).
These complexes were supershifted by addition of antiserum to
STAT5A (data not shown), confirming that the GAS binding complex
contained STAT5A. These data showed that phosphorylated STAT5A, like
STAT4, was capable of forming a DNA binding complex following treatment
with TGF-ß1. We further looked to see whether IL-2-induced
STAT-mediated gene transcription was inhibited by TGF-ß1, by using a
luciferase reporter construct under the control of the STAT binding
site and minimal promoter. We found that TGF-ß1 had no effect on
IL-2-induced transcription of the reporter gene (data not shown). Thus,
the data presented in Figs. 5 and 6 indicate that TGF-ß1 does not
inhibit IL-2-induced activation of the early events in IL-2 signaling,
namely, JAK and STAT phosphorylation and activated STAT binding to DNA.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We thought it important to communicate these findings in light of the
contradictory findings pertaining to the effect of TGF-ß on JAKs and
STATs. For IL-5, TGF-ß1 appears to inhibit phosphorylation of JAK2
and STAT1 in eosinophils 20 . For IFN-, the suppression of
IFN--induced MHC class II gene expression by TGF-ß2 evidently does
not involve the inhibition of phosphorylation of JAKs and STATs but
rather involves the inhibition of CIITA 24, 25 . Studies on the effect
of TGF-ß on IL-2-mediated activation of JAKs and STATs have provided
even more contradictory information. Bright et al. 21 showed the
inhibition of IL-2-induced phosphorylation of JAK1 and STAT5A in Con
A-activated murine T cells but found no effect on phosphorylation of
JAK3 or STAT3. In contrast, Han et al. 22 showed the inhibition of
IL-2-induced phosphorylation of JAK1, JAK3, STAT5A, and STAT3 in Con A
blasts of nonobese diabetic mouse splenocytes. We, however, found no
significant inhibition of phosphorylation of any of these proteins by
TGF-ß1 in human primary T cells and an NK cell line.
It is difficult to offer a clear explanation for the difference between
our data on primary human T cells and the above described studies with
mouse splenocytes. We excluded a trivial explanation, namely, that
TGF-ß1 lacked activity in cells we used, by showing that TGF-ß1
inhibited IFN- production by NK3.3 and primary T cells. We also
confirmed the activity of TGF-ß1 used, by the classical cell growth
inhibition assay. Finally, we also analyzed the effect of TGF-ß1 for
different times and at multiple concentrations of both IL-12 and
TGF-ß1. One obvious, but less than satisfactory, explanation for the
disparate results is the difference in species. It should also be noted
that the above mentioned studies with mouse splenocytes showed partial
and not complete inhibition of phosphorylation of JAKs and STATs. While
the inhibition of IFN- secretion and cell proliferation by TGF-ß1
is also partial, the significance of the partial inhibition of
phosphorylation of JAKs and STATs remains to be determined. Neither
study analyzed DNA binding activity nor activation of transcription.
If TGF-ß1 has no effect on IL-2 and IL-12 early signaling events, how might its actions be effected? TGF-ß signals by binding to a receptor complex composed of type I and type II receptor chains that contain a cytoplasmic serine/threonine kinase domain. The type II receptor is a constitutively active kinase that phosphorylates the type I receptor upon ligand binding, resulting in the activation of the latter, and the propagation of downstream signals 40 . The growing family of SMAD proteins is now known to play an important role in TGF-ß signaling pathway. SMAD2 transiently associates with type I receptor and is phosphorylated in response to TGF-ß 41 . Phosphorylated SMAD2 forms a heteromeric complex with SMAD4, which, like STATs, translocates to the nucleus. The SMAD2-SMAD4 complex regulates gene transcription by interacting with other DNA binding proteins or transcription factors such as FAST1 42 .
One can envision TGF-ß acting by different mechanisms. A distinct
region in the 5' flanking region of CIITA has been identified that
mediates TGF-ß suppression of CIITA 43 . Thus, it is conceivable
that distinct regions in the promoter of IFN- gene could bind SMADs.
Thus, it is possible that SMADs, by binding to IFN- gene promoter,
inhibits IL-12-induced IFN- promoter activity. Whether SMADs are
capable of binding to IFN- gene, directly or in conjunction with
other proteins, and regulating its expression remains to determined.
TGF-ß has also been shown to inhibit myc gene expression 13, 44 and Rb phosphorylation 45, 46 . Further, it up-regulates two cyclin-dependent kinase inhibitors, namely p27kip1 and p15INK4B 47, 48 . These effects have been shown to be the basis for the anti-proliferative action of TGF-ß1. Thus, it is quite possible that the inhibition of IL-2- or IL-12-induced cell proliferation of NK cells and T cells by TGF-ß1 occurs by a direct inhibition of the cell cycle or other factors required for cell proliferation. It is also conceivable that other inhibitory effects of TGF-ß on lymphocytes are brought about partly by its actions on cell proliferation and transit through the cell cycle.
In addition to activation of JAKs and STATs, IL-2 activates a number of
signaling molecules in the Ras/Raf/mitogen-activated protein
kinase (MAPK) and phosphatidylinositol 3' kinase (PI3'K)
pathways. How these pathways impinge on the regulation of genes like
IFN- or cell proliferation is poorly understood. Whether SMADs or
other substrates of the TGF-ß receptor kinase interact with these
pathways is also not known. It is conceivable that TGF-ß inhibits
these pathways, resulting in a partial inhibition of the biological
effects. Thus, several likely mechanisms of inhibition by TGF-ß,
other than the inhibition of JAK-STAT pathway, exist.
The inhibition of JAKs and STATs by TGF-ß1 has been an attractive hypothesis. However, our data argue that the inhibitory effect of TGF-ß1 on IL-12- or IL-2-induced activities is not explained by a direct inhibition of activation of JAKs and STATs in human T cells and NK cells. Clearly, more studies are required to define the exact site of its action. Deciphering the precise mechanism(s) underlying the pleiotropic immunoregulatory effects of TGF-ß will be important and could potentially lead to the development of novel classes of immunosuppressive drugs.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: JAK, Janus kinase; EMSA, electrophoretic mobility shift assay; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; CIITA, class II trans-activator; Ptyr, phosphotyrosine; HRP, horseradish peroxidase; GAS, -activated site; GRR, response region; SMAD, Sma and Mad-related protein; IP, immunoprecipitated; IB, immunoblotted; FAST, forkhead activin signal transducer.
Received for publication March 27, 1998. Accepted for publication November 24, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production and type 1 cytokine responses. Immunity 4:471.[Medline]
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induction of class II MHC gene expression by inhibiting class II transactivator messenger RNA expression. J. Immunol. 158:2065.[Abstract]
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, STAT1, and TGF-ß-regulated expression of the class II transactivator gene. J. Immunol. 160:233.This article has been cited by other articles:
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