1 Division of Molecular and
Cellular Medicine, Thyroid hormone
[L-thyroxine
(T4)] rapidly induced
phosphorylation and nuclear translocation (activation) of
mitogen-activated protein kinase (MAPK) in HeLa and CV-1 cells in the
absence of cytokine or growth factor. A pertussis toxin-sensitive and
guanosine 5'-O-(3-thiotriphosphate)-sensitive
cell surface mechanism responsive to
T4 and
agarose-T4, suggesting a G
protein-coupled receptor, was implicated. Cells depleted of MAPK or
treated with MAPK pathway inhibitors showed reduced activation of MAPK
and of the signal transducer and activator of transcription STAT1
thyroxine; signal transducer and activator of transcription 1 PHYSIOLOGICAL CONCENTRATIONS of thyroid hormone
potentiate the antiviral state induced by homologous interferon
(IFN)- We have recently shown that thyroid hormone promotes tyrosine
phosphorylation and nuclear uptake of STAT1 Materials.
T4,
T3,
3,3',5'-triiodothyronine
(rT3),
3,3',5,5'-tetraiodothyroacetic acid (tetrac),
T4-agarose, protein A-agarose, and
guanosine 5'-O-(3-thiotriphosphate)
(GTP Cell culture and preparation of nuclear fractions.
Confluent HeLa and CV-1 cells grown in 100-mm culture dishes were
treated with 0.25% hormone-depleted fetal bovine serum-containing medium (22) for 48 h. The U3A cell series were grown in the same
serum-supplemented medium with 400 µg/ml G418 added. The total and
free T4 concentrations in this
serum-supplemented medium were 2.3 × 10
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
by
T4; they also showed
reduced T4 potentiation of the
antiviral action of interferon-
(IFN-
).
T4 treatment caused
tyrosine-phosphorylated MAPK-STAT1
nuclear complex formation and
enhanced Ser-727 phosphorylation of STAT1
, in the presence or
absence of IFN-
. STAT1
-deficient cells transfected with STAT1
containing an alanine-for-serine substitution at residue 727 (STAT1
A727) showed minimal
T4-stimulated STAT1
activation.
IFN-
induced the antiviral state in cells containing wild-type
STAT1
(STAT1
wt) or
STAT1
A727;
T4 potentiated IFN-
action in
STAT1
wt cells but not in
STAT1
A727 cells.
T4-directed STAT1
Ser-727
phosphorylation is MAPK mediated and results in potentiated STAT1
activation and enhanced IFN-
activity.
; signal transduction
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(21, 22). Antiviral action of IFN-
requires tyrosine
phosphorylation of the signal transducer and activator of transcription
STAT1
(7). Maximal antiviral activity of IFN-
is obtained when
Ser-727 of STAT1
is phosphorylated (17), perhaps by
mitogen-activated protein kinase (MAPK) (8) or another serine kinase
(45). The 44- and 42-kDa MAPK isoforms extracellular signal-regulated kinase 1 and 2 (ERK1 and ERK2, respectively) are ubiquitously expressed
serine/threonine kinases that are activated by dual-specificity MAPK
kinases (MEK1 and MEK2) in response to diverse agonists (28). A number
of receptor tyrosine kinases, cytokine receptors, and heterotrimeric G proteins have been shown to activate MEK and MAPK
(3, 12).
(20) and have speculated
that the hormone may activate the MAPK pathway to obtain maximal
activation of STAT1
and potentiation of the biological activity of
IFN-
. We report here that thyroid hormone
[L-thyroxine (T4) or
3,5,3'-triiodo-L-thyronine
(T3)] indeed activates the MAPK cascade in HeLa and CV-1 cells, in both the absence and presence of IFN-
. Components of the mechanism by which thyroid hormone activates the MAPK pathway are also described. Because neither HeLa
cells (33) nor CV-1 cells (23) contain functional nuclear thyroid
hormone receptor (TR), the actions of thyroid hormone on kinase
activities in these cell lines are not mediated by TR.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
S) were obtained from Sigma Chemical (St. Louis, MO), and
recombinant human IFN-
was from BioSource International (Camarillo,
CA). Pertussis toxin was obtained from Calbiochem (San Diego, CA).
CGP-41251 was a gift from Novartis Pharma (Basel, Switzerland), and
Lipofectin was obtained from GIBCO BRL (Grand Island, NY). Genistein
was obtained from ICN Biochemicals (Costa Mesa, CA), geldanamycin came
from the Drug Synthesis and Chemistry Branch, National Cancer Institute
(Bethesda, MD), PD-98059 was from Calbiochem (La Jolla, CA), and
U-73122 and U-73343 were obtained from Dr. Robert Smallridge (Mayo
Clinic, Jacksonville, FL). HeLa cells were obtained from the American Type Culture Collection (Manassas, VA), CV-1 cells were from Dr. Paul
M. Yen (National Institutes of Health), and 293T cells were from Dr.
Kevin Pumiglia (Albany Medical College, Albany, NY). U3A,
STAT1
wt, and
STAT1
A727 cells, derived from
human fibroblasts (26, 42), were obtained with the permission of Dr.
George Stark (Cleveland Clinic Foundation, Cleveland, OH) from the
laboratories of Drs. James E. Darnell, Jr., (Rockefeller University,
New York, NY) and Ke Shuai (University of California School of
Medicine, Los Angeles, CA).
[32P]NAD was obtained
from DuPont-NEN (Boston, MA).
11 M and
10
14 M, respectively, and
the free T3 concentration was
below detectable levels (23). Hormone, hormone analogs, IFN-
, and/or
inhibitors were then added for different time periods as indicated.
Stock solutions (10
4 M) of
hormone and analogs were prepared in 0.04 N KOH-4% propylene glycol,
and dilutions were made to final concentrations as indicated. In all
experiments in which T4 was added
to cultures, the total and free T4
concentrations were 10
7 M
and 10
10 M, respectively,
and total and free T3 levels were
below the limits of measurement. The hormone solvent had no effect on
signal transduction studies.
T4-agarose was provided as a
suspension in 0.5 M NaCl containing ~6 mM
T4 and was diluted in culture
medium to a final T4 concentration
of 10
7 M.
Immunoprecipitation and immunoblotting.
After normalization of sample protein content, immunoprecipitation was
performed using polyclonal anti-phosphotyrosine antibody (Transduction
Laboratories, Lexington, KY). After overnight incubation at 4°C
with rocking, protein A-agarose was added and samples were rocked for 1 h at 4°C. After two washes with hypotonic buffer containing 0.2%
NP-40, immunoprecipitates were eluted with 2× sample buffer, and
proteins were separated by discontinuous SDS-PAGE (7.5-9.0%).
Proteins were transferred to Immobilon membranes (Millipore, Bedford,
MA) by electroblotting. After being blocked with 5% milk in
Tris-buffered saline containing 0.1% Tween, membranes were incubated
with 1:1,000 monoclonal anti-MAPK antibody (ERK2) (Transduction Laboratories) or with 1:1,000 monoclonal anti-STAT1 antibody (Transduction Laboratories) overnight. For selected studies, 1:1,000 polyclonal anti-tyrosine/threonine-phosphorylated MAPK antibody (New
England BioLabs, Beverly, MA) was used for immunoblots. In some
experiments (see Fig. 5), nuclear extracts were immunoprecipitated with
monoclonal anti-STAT1
or anti-MAPK antibody (Transduction Laboratories), and the immunoprecipitates were then separated by PAGE
and immunoblotted with antibody to MAPK or STAT1
, respectively. Polyclonal antibodies to Ser-727-phosphorylated STAT1
and to amino
acids 73-93 of TR
1 were generously provided by Dr. David Frank
(Dana-Farber Cancer Institute, Boston, MA) and Dr. William Chin
(Brigham and Women's Hospital, Boston, MA), respectively. The
secondary antibodies were rabbit anti-mouse IgG or goat anti-rabbit IgG
(1:1,000, DAKO, Carpenteria, CA). Immunoblots were visualized by
enhanced chemiluminescence (ECL; Amersham Life Science, Arlington Heights, IL) and quantitated by digital imaging (BioImage, Millipore). Immunoblots shown are representative of two or more experiments.
Cell treatments.
Cells were treated with thyroid hormone or analogs and/or IFN- in
the concentrations indicated. Different concentrations of the protein
kinase C (PKC) inhibitor CGP-41251 (5-100 nM), genistein, a
protein tyrosine kinase (PTK) inhibitor (1-100
µg/ml), or pertussis toxin (20-1,000 ng/ml) were added to
cultures for 70 min, and T4
(10
7 M) was added for the
last 30 min. U-73122, a phospholipase C (PLC) inhibitor (1-10
µM), its inactive analog U-73343, or GTP
S (10
8 to
10
5 M) was added for 60 min, and T4 was added for the last
30 min. Geldanamycin (1-10 µM) or PD-98059 (30 µM) was applied
to cells for 16 h, and 10
7
M T4 was added for the last 30 min. Cells were harvested, and nuclear proteins were prepared as
described. DMSO (0.1%) was the solvent for all inhibitors and had no
effect itself on immunoprecipitation, immunoblotting, or antiviral studies.
Oligonucleotide transfection.
HeLa cells were treated, as described by Glennon et al. (15), with 2.5 µg/ml Lipofectin for 6 h, and sense or antisense oligonucleotides
(Operon Technologies, Alameda, CA) were applied for 48 h in a
concentration of 10 µM. Selected cells from each treatment group were
then exposed to 107 M
T4 for 40 min and subsequently
harvested for preparation of nuclear extracts, immunoprecipitation, and
immunoblotting as described above.
Antiviral studies.
Cells were exposed to IFN- (1.0 IU/ml) in the presence or absence of
T4
(10
7 M) for the last 24 h
of MAPK antisense oligonucleotide transfection. In studies with MAPK
pathway inhibitors, cells were treated with T4, with or without geldanamycin
or PD-98059, for 24 h; the medium was then replaced, and cells were
treated with IFN-
(1.0 IU/ml) for 24 h.
STAT1
wt and
STAT1
A727 cells were treated
with IFN-
, with or without T4,
for 24 h. After these various treatments, the cells were infected with
vesicular stomatitis virus, and an antiviral plaque assay was performed
as described previously (22), with results expressed in plaque-forming
units (pfu) per milliliter. In our antiviral studies, a maximal effect
of IFN-
is seen at a cytokine concentration of 1,000 IU/ml, but
T4 potentiation of IFN-
action
is greatest at submaximal IFN-
concentrations [1-10 IU/ml
(22)]. The plaque assay method does not allow accurate measurement of virus yield below
104 pfu/ml. One-way ANOVA was used
to determine statistical significance.
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RESULTS |
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Thyroxine and analogs cause tyrosine phosphorylation and nuclear
translocation of MAPK.
Initial studies were performed with
T4 alone, in the absence of
IFN-. Confluent HeLa cells were treated with 0.25% thyroid hormone-depleted serum-supplemented medium for 2 days, and
T4 (10
7 M total,
10
10 M free) was then added
to cultures for 10-60 min. This concentration of
T4 is physiological. Cells were
harvested, and nuclear extracts were immunoprecipitated with
anti-phosphotyrosine antibody. Immunoprecipitated proteins were eluted
from protein A-agarose, separated by electrophoresis, and
immunoblotted with anti-MAPK antibody. Increased nuclear content of
tyrosine-phosphorylated MAPK was detected at 10 min after exposure to
T4 (Fig.
1A).
This transient effect was maximal at 30-40 min and significantly
reduced or absent at 60 min. In four experiments, the increase in band
density with T4 addition for 30 min, compared with a control sample without hormone, was 25 ± 8-fold (means ± SE). Nuclear tyrosine-phosphorylated MAPK was also
detected by immunoblotting of nuclear samples with
anti-tyrosine/threonine-phosphorylated MAPK antibody, as shown in Fig.
1B. In the study shown, accumulation of nuclear activated MAPK was maximal at 30 min, and there was a
34-fold increase in combined band intensities of ERK1 and ERK2.
|
|
Cell surface action of T4.
The T4 analog tetrac blocks
1)
T4 potentiation of IFN--induced
human leukocyte antigen-DR (HLA-DR) expression (20),
2) T4 potentiation of IFN-
-induced
antiviral activity (21, 23), and 3)
T4-induced activation of STAT1
(20). Tetrac also partially inhibited the effect of
T4 on tyrosine phosphorylation and
nuclear translocation of MAPK, as shown in Fig.
2B
(lanes
4 and
5, compared with
lane
2). We previously showed that tetrac
inhibits binding of T4 to isolated
human erythrocyte membranes (10) and therefore postulated that tetrac
blocks T4 activation of
signal-transducing proteins by inhibition of
T4 interaction at the cell
surface. To further study this possibility, we treated HeLa cells with T4-agarose. In Fig.
2C, we show that
T4,
T4-agarose, and
T4-agarose clarified with three
PBS washes all stimulated tyrosine phosphorylation and nuclear
translocation of MAPK (lanes
3-5),
whereas protein A-agarose had no effect on MAPK activation
(lane
2). These findings further support
the action of T4 at the cell
membrane as an enhancer of signal transduction. We have also conducted
these studies in CV-1 cells, which like HeLa cells lack a traditional
TR (23), and have obtained similar results (not shown).
Involvement of a G protein-coupled mechanism of hormone action.
Della Rocca et al. (11) reported rapid activation of MAPK by
-adrenergic agonists via pathways mediated by the G proteins Gi and
Gq. To define the possible role of
a G protein-coupled mechanism at the cell surface in the action of
T4, GTP
S was added to HeLa
cells for 70 min, and T4 was added
for the last 30 min of GTP
S treatment. Nuclear fractions were
prepared and immunoblotted with anti-phosphorylated MAPK antibody, and
the results are shown in Fig.
3A. There
is T4-induced phosphorylation and
nuclear accumulation of MAPK and dose-dependent reduction of the
T4 effect by GTP
S. HeLa cells
were also treated with pertussis toxin (20-1,000 ng/ml) for 60 min
and with T4 for 30 min;
immunoblots with phosphorylated MAPK antibody showed a dose-dependent
reduction of the T4 effect by
pertussis toxin (Fig. 3B). In the
absence of T4, pertussis toxin did
not alter MAPK phosphorylation (results not shown). These results
suggest a contribution of a pertussis-toxin-inhibitable GTP-binding
protein (Gi or
Go) to the
T4 effect and raise the possibility that thyroid hormone binds to a
Gi protein-coupled receptor (GPCR)
at the cell membrane.
|
Contributions of PKC, PTK, and PLC to the thyroxine effect.
We previously reported that T4
potentiation of IFN--induced HLA-DR expression and antiviral
activity is dependent on activities of PKC and PTK (20, 23).
Investigation of the effects of kinase inhibitors on
T4 activation of MAPK was
therefore undertaken. Genistein (1-100 µg/ml), an inhibitor of
PTK activity (30), blocked the action of
T4 on MAPK phosphorylation and
nuclear translocation (Fig.
4A).
CGP-41251, an inhibitor of calcium-dependent PKC activity [inhibits PKC
, -
I,
-
II, and -
with
IC50 values of 24, 17, 32, and 18 nM, respectively (25)], also blocked
T4-induced tyrosine phosphorylation of MAPK in a concentration-dependent manner (Fig. 4B). In Fig.
4C, genistein and CGP-41251 are again
shown to inhibit the effect of T4
on MAPK phosphorylation and nuclear translocation in nuclear samples
immunoblotted with antibody to tyrosine/threonine-phosphorylated MAPK.
The two bands representing ERK1 (44 kDa) and ERK2 (42 kDa) are
similarly affected by T4 and the
inhibitors. The effect of the PLC inhibitor U-73122 (35) and an
inactive analog, U-73343, on T4
activation of MAPK was also examined. Figure
4D shows activation of MAPK by
T4 in HeLa cells and inhibition of
this MAPK activation by U-73122 but not by U-73343. Similar results
were obtained with 293T cells, which contain TR, as indicated by
immunoblotting with antibody to TR
(results not shown).
|
Coimmunoprecipitation of MAPK and STAT1.
David et al. (8) described coimmunoprecipitation of MAPK and STAT1
in extracts of cells treated with IFN-
. We therefore tested the
possibility that a direct interaction between MAPK and STAT1
could
be detected in cells treated with
T4, alone or with IFN-
. Nuclear
extracts were immunoprecipitated with anti-MAPK antibody, and the
solubilized immunoprecipitates were subjected to electrophoresis and
immunoblotted with anti-STAT1
. Nuclear STAT1
appeared in MAPK
immunoprecipitates in cells treated with T4 alone
(10
7 M), as shown in Fig.
5A. In the
study shown in Fig. 5B, cells were
treated with T4 with or without
IFN-
(1-100 IU/ml) for 30 min. The antibody order was reversed,
and samples of nuclear extracts were immunoprecipitated with
anti-STAT1
antibody and the precipitates were immunoblotted with
anti-MAPK antibody. Both T4 and
IFN-
separately caused nuclear complexing of STAT1
and MAPK, as
reported previously with IFN-
(8). At each concentration of IFN-
, T4 enhanced the cytokine effect.
Additional studies using an antibody to Ser-727-phosphorylated STAT1
(13) demonstrated increased Ser-727 phosphorylation of STAT1
by
T4 or IFN-
and potentiation of
the cytokine effect by thyroid hormone (Fig.
5C).
T4 and IFN-
(1.0 IU/ml), when
applied separately to cells, caused detectable Ser-727 phosphorylation.
With addition of hormone together with a low concentration of cytokine
(1 IU/ml), there was enhancement of IFN-
-induced Ser-727
phosphorylation by T4. With a
higher IFN-
concentration, there was no further enhancement of the
IFN-
effect by T4.
|
Effect of MAPK pathway inhibition.
Studies were undertaken to characterize more proximal steps in the MAPK
cascade that might contribute to the
T4 effect on activation of MAPK
and STAT1 and potentiation of IFN-
action. Geldanamycin, a MAPK
pathway inhibitor that incompletely depletes cellular content of Raf-1
(32, 36), partially inhibited
T4-stimulated nuclear uptake of
tyrosine-phosphorylated MAPK (Fig.
6A,
top, lane
3) and tyrosine-phosphorylated
STAT1
(Fig. 6A,
bottom,
lane 3). Similar findings were obtained
with an inhibitor of MEK, PD-98059 (27), and are shown in Fig.
6A
(lanes
4-6);
there was reduction or complete inhibition of
T4-stimulated activation of MAPK
and STAT1
by PD-98059 (lane
6,
top and
bottom).
|
Effect of MAPK antisense oligonucleotide transfection.
To further examine the role of the MAPK pathway in
T4 potentiation of IFN- action,
we reduced the HeLa cell content of MAPK by antisense oligonucleotide
transfection (15). Absence of activated MAPK in nuclear fractions of
cells treated with the antisense oligonucleotide is shown in Fig.
7A
(top,
lane
7), and there is little tyrosine
phosphorylation or nuclear translocation of MAPK in antisense-treated
cells exposed to T4
(top,
lane
8), particularly compared with
levels in cells treated with Lipofectin alone
(lane 4). In the same MAPK-depleted cells,
there was a reduction in T4-stimulated nuclear
translocation of tyrosine-phosphorylated STAT1
, compared with
findings in HeLa cells not exposed to the MAPK antisense
oligonucleotide (Fig. 7A,
bottom,
lane
8 compared with
lanes
2 and
4). This finding suggests that the
presence of stimulable MAPK is a requirement for optimal activation and
nuclear translocation of STAT1
. In cells treated with sense
oligonucleotide, there was more activated MAPK in nuclei of cells not
exposed to T4 than in cells
exposed to the hormone (Fig. 7A,
top,
lanes
5 and
6). The basal increase in MAPK and
reduced activity of MAPK in response to an inducer in these cells have
been described by other investigators in a different cell line (29). To
test the effect of cellular MAPK depletion on
T4 potentiation of IFN-
action,
antiviral studies were also conducted on cells transfected with MAPK
antisense or sense oligonucleotide or with Lipofectin alone.
Significant T4 potentiation of the
antiviral action of IFN-
(P < 0.05), shown by the vertical arrows in Fig.
7B, was demonstrated in untreated
(control) cells and cells treated with sense oligonucleotide or
Lipofectin alone but was diminished in MAPK-depleted cells. In the
latter cells, however, the antiviral response to IFN-
(1.0 IU/ml) in
the absence of T4 was not
diminished. Taken together, the results of experiments with MAPK
pathway inhibition and cellular depletion of MAPK provide evidence that
T4-induced activation and nuclear
translocation of MAPK and STAT1
, and potentiation of the action of
IFN-
, require activity of Raf-1 and MEK, as well as MAPK. These
antiviral experiments with MAPK depletion and MAPK pathway inhibitors
were conducted with submaximal concentrations of IFN-
.
|
T4 potentiation of IFN-
action requires Ser-727 on STAT1
.
Maximal antiviral activity of IFN-
requires the presence of a serine
at position 727 of STAT1
(17). It has been suggested that MAPK may
catalyze the Ser-727 phosphorylation of STAT1
(8, 42), although more
recent studies suggest that the Ras-MAPK pathway is not involved, at
least in IFN-
-induced serine
phosphorylation (45). Because we have demonstrated
T4-induced nuclear accumulation of
Ser-727-phosphorylated STAT1
(Fig.
5C), we studied whether T4 potentiation of IFN-
action
would be seen in cells lacking serine at position 727. U3A cells lack
STAT1
(26, 42) and on exposure to
T4 showed no nuclear accumulation
of that protein (Fig.
8A,
top). U3A cells with reconstituted
wild-type STAT1
(STAT1
wt)
showed stimulation of STAT1
tyrosine phosphorylation and nuclear
translocation by T4, whereas U3A
cells containing STAT1
with an alanine-for-serine substitution at
position 727 (STAT1
A727)
showed diminished activation of that protein (Fig. 8A,
top). In the same samples, nuclear
accumulation of tyrosine-phosphorylated MAPK appeared to be more
intense in U3A cells and
STAT1
A727 cells than in the
STAT1
wt cells (Fig.
8A,
bottom), demonstrating that activation of MAPK does not require the presence of STAT1
, whereas activation of STAT1
does seem to require the presence of functional MAPK (see Figs. 6 and 7).
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DISCUSSION |
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The present observations support a novel role for thyroid hormone as a
modulator of signal transduction. We used a cytokine, IFN-, to
demonstrate the biological relevance of thyroid hormone action on two
signal transduction pathways. We propose that the enhancement by
thyroid hormone of IFN-
-induced antiviral activity depends on
effects of the hormone on the MAPK cascade and, as a result, on the
STAT1
pathway that is activated by the cytokine. The consequence of
the action of thyroid hormone on MAPK phosphorylation and nuclear
translocation is viewed by us to be Ser-727 phosphorylation of the
STAT1
dimer that results in increased binding of the STAT complex to
one or more IFN-
-responsive genes. Activated STAT1
is a critical
phosphoprotein in the transduction of IFN-
and IFN-
/
signals
(7). Activation of STAT1
by IFN-
requires Tyr-701 phosphorylation
by Janus kinases (JAK1 and JAK2); maximal transcriptional activity of
STAT1
requires, in addition, phosphorylation of Ser-727
(17, 42). MAPK has been implicated in IFN-
/
signal transduction, specifically in the Ser-727 phosphorylation of STAT1
(8) and also in Ser-727 phosphorylation of another member of the
STAT family, STAT3 (5).
The ability of the two MAPK pathway inhibitors, geldanamycin and
PD-98059, to reduce activation of MAPK and STAT1 and to block
thyroid hormone potentiation of the antiviral action of IFN-
further
supports our hypothesis that the mechanism of
T4 in this potentiation involves
the MAPK pathway, principally at the levels of MEK and MAPK.
Geldanamycin incompletely depletes cellular content of Raf-1 in MCF7
cells (32) and HeLa cells (36), whereas PD-98059 is regarded as a
specific inhibitor of MEK activity at the concentrations we used (4,
27). Tyrosine phosphorylation of STAT1
is an integral part of the
IFN-
-stimulated JAK-STAT pathway. In the absence of IFN-
,
however, the means by which T4
caused tyrosine phosphorylation of STAT1
required clarification. The
results obtained with PD-98059 presented in Fig. 6 suggest that, under
the direction of thyroid hormone, MEK tyrosine phosphorylates STAT1
,
in addition to activating its traditional substrate, MAPK. MEK is a
dual-specificity tyrosine-threonine kinase. Phosphorylation of
threonines at positions 699 and 704 of STAT1
(44) may, as described
by Cobb and Goldsmith (6), provide an environment suitable for
phosphorylation of Tyr-701 by MEK. Our results do not exclude the
possibility that an unidentified MEK-dependent tyrosine kinase is
responsible for T4-directed
tyrosine phosphorylation of STAT1
. Depletion of cellular MAPK by
antisense oligonucleotide transfection also confirmed that MAPK is
required for T4-induced activation
of STAT1
and for T4
potentiation of the antiviral activity of IFN-
.
Fukuda et al. (14) recently reported that the transport of MAPK from cytoplasm to nucleus requires dissociation of the MAPK-MEK complex. It is possible that thyroid hormone may also act at this putative complex to enhance nuclear transfer of MAPK. It should be noted that tyrosine phosphorylation (activation) of MAPK in the present studies was documented by both anti-phosphotyrosine antibody immunoprecipitation with subsequent probing of the immunoprecipitate with anti-MAPK antibody and by the use of antibody to phosphorylated MAPK, which indicated involvement of both ERK1 and ERK2 in the hormone effect. Gorenne et al. (16) recently demonstrated correlation of two measurements of MAPK activation, 1) immunoblotting with antibody to tyrosine/threonine-phosphorylated MAPK and 2) direct measurement of MAPK activity by phosphorylation of myelin basic protein.
The initial step in the mechanism by which thyroid hormone acts on
signal transduction is incompletely understood. Several features are
clear, however. Because agarose-T4
is as effective as T4 in
activating MAPK and STAT1, hormone action must begin at the cell
surface. Furthermore, the hormone effect is inhibited by tetrac, an
iodothyronine analog that, itself, is not a kinase activator but does
inhibit the binding of T4 to human
erythrocyte membranes (10). Consistent with this evidence that hormone
action requires a putative receptor on the plasma membrane are the
observations presented here that
T4 action on signal transduction
is pertussis toxin and GTP
S sensitive. Thus the initial step in the
mechanism is interaction of thyroid hormone with a GPCR. We previously
described a Gi protein in
erythrocyte membranes that may be involved in hormone action (9, 38),
but a GPCR responsive to thyroid hormone has not been previously
reported in nucleated cells (39). By nondenaturing PAGE of
octylglucoside-solubilized plasma membranes, we recently identified two
proteins that bind radiolabeled T4 and are candidate GPCRs (M. R. Deziel, F. B. Davis, and P. J. Davis,
unpublished observations).
PKC has been shown to activate Raf-1 directly (18), a step that is
early in the MAPK cascade (Raf-1/MEK/MAPK). The fact that CGP-41251, an
inhibitor of traditional PKC isoform activities (PKC
,
-
I,
-
II, and -
), prevented the
action of thyroid hormone in our model is consistent with an effect of
T4 at the level of Raf-1, although
our findings also raise the possibility that activated PKC can mediate
the action of T4 through the
phosphorylation of MEK, as described by others (34), thus bypassing
Raf-1. With the use of CGP-41251, we have effectively ruled out a
contribution of calcium-independent, diacylglycerol-dependent novel
PKCs and atypical PKCs, as described by Schönwasser et al. (31),
some of which phosphorylate Raf-1 without stimulating its kinase
activity. We previously reported that physiological concentrations of
T4 stimulate erythrocyte cytosol
PKC activity (19), also supporting a role for PKC stimulation in the
T4 effects we describe in this report. We have elsewhere demonstrated a role for PKC in thyroid hormone potentiation of both antiviral (23) and immunomodulatory (20)
actions of IFN-
. A role for PLC in iodothyronine action on the MAPK
cascade is suggested by results of our studies with the aminosteroid
U-73122. That is, hormone activation of PKC (and subsequently of the
MAPK pathway) depends on diacylglycerol liberated by PLC.
The proposed sequence of events in the signal transduction pathways
acted on by thyroid hormone is shown in Fig.
9. Hormone binding at the cell membrane is
followed serially by activation of PLC, PKC, Raf-1, MEK, and MAPK. The
possibility that Ras may be involved in the sequence is included in
Fig. 9. Although pertussis toxin-sensitive GPCRs have not previously
been described to activate Ras through PLC and PKC (39, 40), we have
found that thyroid hormone poorly activates MAPK in a dominant negative
Ras model (J. Gordinier, F. B. Davis, and P. J. Davis, unpublished
observations). Thus Raf-1 may be phosphorylated by PKC in the thyroid
hormone-activated cell or PKC may act on Ras (24), thus activating
Raf-1.
|
Apparent from the present studies are not only the promotion by
T4 of the nuclear uptake of
activated STAT1 and MAPK but also the recovery of MAPK from nuclear
immunoprecipitates made with anti-STAT1
antibody and of STAT1
from anti-MAPK immunoprecipitates. Thus the nuclear fractions in
T4-treated cells contain a
STAT1
-MAPK complex. Association of MAPK and STAT1
has been
described by others in response to IFN-
/
(8). We have not
detected this complex in cytosol of hormone-treated cells. Ostensibly,
the complex reflects the action of MAPK in phosphorylating Ser-727 of
STAT1
, as suggested by David et al. (8). Documentation of thyroid hormone-potentiated phosphorylation of Ser-727 of STAT1
in the presence of IFN-
is shown in Fig.
5C. Thus the complexing of STAT1
and MAPK under the influence of T4
is not a casual event but is associated with phosphorylation of a
specific residue of STAT1
. Our studies with the U3A cell series
revealed that, in the absence of STAT1
, activated MAPK did
translocate to the nucleus in the presence of
T4, whereas
T4-induced nuclear translocation of STAT1
was suppressed in the absence of MAPK in antisense
oligonucleotide-treated cells.
In the absence of IFN-, thyroid hormone does not induce the
antiviral state (22), so that activation of the MAPK pathway by the
hormone is insufficient to initiate transcription of antiviral proteins. This is not surprising, since it is the STAT1
pathway that
is primarily involved in transducing the IFN signal for the antiviral
state and MAPK activity has been shown by others to be facilitative
(17, 42). We postulate that there is a biological role for thyroid
hormone potentiation in the presence of low levels of IFN-
; in fact,
we have found that T4 promotes the
antiviral action of IFN-
at 0.1 IU/ml, a concentration that in
itself is not antiviral (22). Thus, through enhanced activation of MAPK and STAT1
, the hormone is able to convert an ineffective level of
IFN-
into an effective concentration.
It is possible that one or more actions of thyroid hormone on kinase
cascades are representative of a newly recognized mechanism of hormone
signaling, one that 1,25-dihydroxyvitamin
D3 also utilizes; the vitamin has
been reported by others to stimulate activity and translocation of
protein kinases via a nongenomic mechanism in acute promyelocytic NB4
cells (2). Other hormones that activate kinases in the MAPK cascade
include gonadotropin-releasing hormone (37), norepinephrine (43), and
17-estradiol (41). Inhibition of MEK has been shown to decrease
growth hormone-stimulated activation of STAT5 (27) and to decrease
insulin stimulation of Ser-727 phosphorylation of STAT3 (4). Thus
hormones that have important actions via nuclear receptors, as well as
those that act primarily at the cell membrane, can nongenomically alter
signal-transducing kinase activities.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank the Drug Synthesis and Chemistry Branch, Developmental
Therapeutics Program, Division of Cancer Treatment, National Cancer
Institute (Bethesda, MD) for the provision of geldanamycin, Drs. George
Stark (Cleveland, OH), James E. Darnell, Jr., (New York, NY), and Ke
Shuai (Los Angeles, CA) for provision of U3A, STAT1wt, and
STAT1
A727 cells, Dr. David
Frank (Boston, MA) for the antibody to Ser-727-phosphorylated STAT1
,
and Dr. William Chin (Boston, MA) for the TR
1 antibody.
![]() |
FOOTNOTES |
---|
This work was supported in part by funding from the Office of Research Development, Medical Research Service, Department of Veterans Affairs (to P. J. Davis) and by a grant from the Candace King Weir Foundation.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: P. J. Davis, Dept. of Medicine A-57, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208 (E-mail: pjdavis{at}albany.net).
Received 20 October 1998; accepted in final form 19 January 1999.
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