Protein synthesis-dependent potentiation by thyroxine of
antiviral activity of interferon-
Hung-Yun
Lin,
Paul M.
Yen,
Faith B.
Davis, and
Paul J.
Davis
Department of Medicine, Albany Medical College and Stratton Veterans
Affairs Medical Center, Albany, New York 12208; and Division of
Genetics, Department of Medicine, Brigham and Women's Hospital and
Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
We have studied
the prenuclear signal transduction pathway by which thyroid hormone
potentiates the antiviral activity of human interferon-
(IFN-
) in
HeLa cells, which are deficient in thyroid hormone receptor (TR). The
action of thyroid hormone was compared with that of milrinone, which
has structural homologies with thyroid hormone.
L-Thyroxine
(T4),
3,5,3'-L-triiodothyronine (T3), and milrinone enhanced the
antiviral activity of IFN-
up to 100-fold, a potentiation blocked by
cycloheximide. The 5'-deiodinase inhibitor
6-n-propyl-2-thiouracil did not block
the T4 effect. 3,3',5,5'-Tetraiodothyroacetic acid prevented the effect of
T4 but not of milrinone. The
effects of T4 and milrinone were
blocked by inhibitors of protein kinases C (PKC) and A (PKA) and
restored by PKC and PKA agonists; only the effect of
T4 was blocked by genistein, a
tyrosine kinase inhibitor. In separate models, milrinone was shown not
to interact with nuclear TR-
.
T4 potentiation of the antiviral
activity of IFN-
requires PKC, PKA, and tyrosine kinase activities
but not traditional TR.
thyroid hormone action; protein kinase C; protein kinase A; tyrosine kinase
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INTRODUCTION |
WE HAVE DEMONSTRATED that iodothyronines potentiate the
antiviral activity of recombinant human interferon-
(IFN-
) by up to 140-fold in HeLa cells and human fibroblasts (13). This thyroid hormone potentiation occurs by two mechanisms, one protein synthesis dependent and the other postnuclear or nongenomic and independent of
protein synthesis (11). The nongenomic, or protein
synthesis-independent effect of thyroid hormone has been shown to
involve activities of both protein kinase C (PKC) and adenosine
3',5'-cyclic monophosphate (cAMP)-dependent protein kinase
(PKA) (12). The possible contributions of these kinases to the protein
synthesis-dependent actions of thyroid hormone have not been explored.
In early studies of this latter effect, a physiological concentration
of L-thyroxine
(T4) (13) has been shown to be
more effective than a physiological concentration of
3,5,3'-L-triiodothyronine
(T3), and
3,3',5'-L-triiodothyronine [reverse T3
(rT3)] has also been found
to be active (11). This action of
T4 is blocked by
3,3',5,5'-tetraiodothyroacetic acid (TETRAC), whereas the
nongenomic effect of T4 is not
blocked by TETRAC (11).
Milrinone is a bipyridine with cardiac inotropic properties (1) that
also stimulates bone turnover and calcium release from bone (10). Its
mechanisms of action include inhibition of cyclic nucleotide
phosphodiesterase activity (7) and stimulation of tissue-specific
membrane calcium- and magnesium-dependent adenosinetriphosphatase (Ca2+-ATPase) activity (14, 23).
We have reported that milrinone has structural homologies with thyroid
hormone (14) and bioactivities that overlap those of thyroid hormone,
including stimulation of myocardial membrane (14) and skeletal muscle
sarcoplasmic reticulum (23)
Ca2+-ATPase activities and binding
to the thyroid hormone binding protein, transthyretin (5).
Because of structural similarities between milrinone and thyroid
hormone, we have used milrinone as a tool, along with selected PKC and
PKA agonists and PKC, PKA, and tyrosine kinase antagonists, to identify
possible signal transduction pathways that may contribute to the
enhancement of IFN-
's antiviral activity by both thyroid hormone
and milrinone. We have also compared the effects of milrinone with
those of T3 in traditional thyroid
hormone receptor-
(TR-
) binding and cotransfection studies to
further contrast the bipyridine's effects with those of thyroid
hormone.
 |
MATERIALS AND METHODS |
Reagents.
Recombinant human IFN-
(>1 × 107 U/mg) was obtained from
BioSource International (Camarillo, CA) and was used for all studies. T4,
T3,
3,3',5-triiodothyroacetic acid (TRIAC),
6-n-propyl-2-thiouracil (PTU),
cycloheximide, milrinone, amrinone, 3-isobutyl-1-methylxanthine (IBMX),
phorbol 12-myristate 13-acetate (PMA), and 8-bromo-cAMP (8-BrcAMP) were
obtained from Sigma Chemical (St. Louis, MO). TETRAC was obtained in
pure form except for minimal salt content from Henning Berlin (G. Weickgenannt, personal communication); there is no thyroid hormone or
TRIAC in this product. KT-5720, a PKA inhibitor with an inhibitor
constant of 6 × 10
8 M
(9), was obtained from Kamiya Biomedical (Thousand Oaks, CA), and
CGP-41251, a PKC inhibitor (3), was kindly provided by Ciba-Geigy
(Basel, Switzerland). The tyrosine kinase inhibitor genistein (17) was
obtained from ICN Biochemicals (Costa Mesa, CA).
Cell cultures.
HeLa cell cultures were grown and maintained in Dulbecco's modified
Eagle's medium (DMEM), supplemented with 10% fetal bovine serum
(FBS), as described previously (13). HeLa cells are deficient in TR
(19). The serum was depleted of thyroid hormone by treatment with an
anion exchange resin (AG1-X8, Bio-Rad Laboratories, Richmond, CA),
according to the method of Weinstein et al. (24). As a result, the free
and total T4 concentrations in
hormone-depleted serum-supplemented medium (SSM), measured by analog
radioimmunoassay and chemiluminescent assay, were 0.4 × 10
12 and 0.9 × 10
9 M, respectively, and
the total T3 concentration was
<10
10 M (13). There was
no free T3 measurable in
hormone-depleted serum by a commercial assay (Laboratory Corporation of
America). Murine fibroblasts (L-929) were grown and maintained in
FBS-supplemented minimal essential medium (MEM); these cells were used
for assay of antiviral activity (see below).
Virus culture and quantitation of antiviral state.
Confluent monolayers of HeLa cells
(105 cells/well) were grown in
24-well trays at 37°C and refed with fresh medium containing thyroid hormone-depleted FBS (10%) for 24 h before addition of IFN-
. Cultures then received 1.0 IU/ml IFN-
or diluent in medium (control) for an additional 24 h at 37°C, followed by viral
challenge. A plaque-purified preparation of vesicular stomatitis virus
(VSV, Indiana serotype) was maintained for determination of antiviral state and IFN-
titration. Antiviral state was measured by infecting control and IFN-
-treated cells with VSV at an input multiplicity of
infection of 10 plaque-forming units (pfu) per cell; virus yield
(pfu/ml) was quantitated by plaque assay in L-929 cells according to
methods previously described (13).
Antiviral activity of IFN-
and effect of thyroid
hormone and bipyridines.
Stock solutions (10
4 M) of
T4 and analogs in 4% propylene
glycol-0.04 N KOH and 10
3 M
preparations of milrinone and amrinone in dimethyl sulfoxide (DMSO)
were diluted 1:100 or more in SSM as needed. IBMX was dissolved in
100% ethanol in a stock concentration of 5 × 10
2 M and applied to cells
in a final concentration of 5 × 10
4 M. PTU was dissolved
directly in SSM in a concentration of
10
3 M. Control incubations
contained diluent in appropriate concentrations. Diluents used had no
antiviral effect when applied alone in SSM to cells and did not alter
antiviral response to IFN-
. HeLa cells were incubated with
T4 or bipyridine for either a 24-h
preincubation before 24-h treatment with IFN-
(1.0 IU/ml) or the
last 4 h of a 24-h IFN-
exposure. Antiviral assays were then
performed. Results are shown as means ± SE of two or more
experiments. One-way analysis of variance (ANOVA) was used to determine
statistical significance of hormone potentiation and inhibition of that
potentiation.
Effect of cycloheximide and protein kinase agonists and antagonists
on potentiation of the antiviral activity of IFN-
.
In selected studies, cycloheximide (CHX, 5 µg/ml) was added to
cultured cells with or without T4
or milrinone for 24 h before IFN-
treatment in the preincubation
paradigm described above. The medium containing CHX and hormone or
bipyridine was then removed and replaced with fresh medium containing
1.0 IU/ml IFN-
for 24 h, after which the antiviral state was
determined. Under these conditions, protein synthesis, measured by
[35S]methionine
incorporation into proteins and subsequent counting of trichloroacetic
acid precipitates, was inhibited by 63% in the cells exposed to CHX.
Because CHX was removed before addition of medium containing IFN-
,
there was no inhibition of the IFN's antiviral effect during the
subsequent 24-h incubation.
The effect of kinase agonists and antagonists was similarly tested by
adding those reagents to the culture medium during the 24-h
preincubation with thyroid hormone or milrinone. 8-BrcAMP was dissolved
in distilled water. The agonist PMA and kinase antagonists were
dissolved in DMSO so that a final DMSO concentration of 0.1% or less
was attained. As in the studies with CHX, the media were changed before
the addition of IFN-
to the cells, so that agonists and/or
inhibitors were not present during IFN-
treatment.
T3 binding studies using nuclear
TR-
.
A previously described expression vector (16) containing human TR-
cDNA was subcloned into the Nde I and
BamH I sites of pET16b vector
containing sequences encoding histidine residues in the leader sequence
(Novagen; gift of Dr. Kevin Petty, University of Texas Southwestern).
TR was then expressed in BL21(DE3) pLys S Escherichia
coli cells, and the expressed protein was purified using nickel-bound resin (Qiagen, Chatsworth, CA) according to the
manufacturer's instructions. The molecular weight and purity of
isolated protein were verified by electrophoresis and Coomassie blue
staining on a 10% polyacrylamide gel.
T3 binding studies were performed
as previously described (21).
Cotransfection studies.
Previously described cDNA clones of rat TR-
in pSG5 (20) or TR-
in pCDNA (27) were used in the cotransfection experiments. A previously
described reporter plasmid containing the chicken lysozyme thyroid
hormone response element (TRE) F2 and luciferase cDNA was also used
(26). CV-1 cells were grown in DMEM containing 10% fetal calf serum.
The serum for these studies was depleted of
T3 and steroid hormones by
charcoal stripping for 12 h at 4°C and constant mixing with 5%
(wt/vol) AG1-X8 resin twice for 12 h at 4°C before ultrafiltration.
The cells were transfected with expression (0.1 µg) and reporter (1.7 µg) plasmids as well as respiratory syncytial virus (RSV)
-galactosidase control plasmid (1 µg) in 3.5-cm
plates using the calcium phosphate precipitation method (26). Cells
were grown for 48 h in the absence or presence of various
concentrations of the T3 or
milrinone and harvested. Cell extracts were then analyzed for both
luciferase and
-galactosidase activity (26). Luciferase activity was
normalized to
-galactosidase activity, and the magnitude of
induction relative to basal transcription was calculated, with onefold
basal equaling transcription activity with empty expression vector
alone in the absence of ligand.
 |
RESULTS |
Effect of thyroid hormone and bipyridines on antiviral activity of
IFN-
in HeLa cells.
The potentiating effects of pretreatment with graded concentrations of
T4 and
T3 on the antiviral action of
IFN-
subsequently applied to HeLa cells are shown in Fig.
1. There is a 10-fold, or one log, decrease
in virus yield in cells treated with IFN-
(1.0 IU/ml) compared with
untreated (control) cells. Although 10
11 to
10
10 M concentrations of
both hormones were ineffective, a progressive increase in
T4 and
T3 concentration to
10
7 M produced 54- and
42-fold increases, respectively, in antiviral activity
(P < 0.01), shown as decreases in
virus yield compared with the yield with IFN-
alone. The
hormone-depleted SSM contained <10
9 M total
T4 and
<10
10 M total
T3. We regard
10
7 M total
T4 and
10
9 M total
T3 as physiological
concentrations. Thus our studies show that, at physiological
concentrations, T4 is more potent than T3 in this intact HeLa cell
antiviral model. To document that the conversion of
T4 to
T3 is not contributing to the
T4 effect, PTU was preincubated
with T4. PTU neither altered the antiviral response to IFN-
nor had any effect on
T4 potentiation as shown in Fig.
2.

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Fig. 1.
Concentration-dependent potentiation by
L-thyroxine
(T4) and
3,5,3'-L-triiodothyronine
(T3) of antiviral effect of
interferon- (IFN- ). In this and subsequent antiviral figures,
open bar represents control virus yield and filled bar represents
antiviral effect of IFN- (1.0 IU/ml). Horizontal arrow below graph
also indicates samples that received IFN- .
T4 or
T3
(10 11 to
10 7 M) was added to cells
for a 24-h preincubation before 24-h IFN- treatment as described in
MATERIALS AND METHODS. In all studies, there was a complete change of medium between preincubation and IFN-
treatment periods. Statistical significance of potentiation by
T4 and
T3 at each concentration was
measured by 1-way analysis of variance (ANOVA), comparing virus yield
with that in samples treated with IFN- alone. pfu, Plaque-forming
units. Levels of significance:
* P < 0.05;
** P < 0.01.
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Fig. 2.
Lack of effect of
6-n-propyl-2-thiouracil (PTU) on
T4 potentiation of IFN- 's
antiviral action. PTU did not inhibit antiviral action of IFN- (1.0 IU/ml) and did not inhibit T4
potentiation of antiviral effect.
** P < 0.01, significant
effect of T4 pretreatment on virus
yield in IFN- -treated cells.
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The effect of T4, milrinone, and
amrinone on the antiviral action of IFN-
is shown in Fig.
3. Cells preincubated with
10
7 M
T4 for 24 h before IFN-
exposure showed a 71-fold reduction in virus yield, compared with the
yield in cells treated with IFN-
alone. A similar effect was seen
when T4 was added to cells during
only the last 4 h of the 24-h IFN-
incubation (4-h coincubation). Milrinone (10
7 to
10
5 M) enhanced the
antiviral effect of IFN-
from 7- to 41-fold, respectively, when
added to HeLa cells in the 24-h pretreatment paradigm (Fig. 3) but had
no effect during a 4-h coincubation with IFN-
. Neither
T4 nor milrinone had antiviral
activity in the absence of IFN-
(results not shown). Amrinone
(10
7 M) did not enhance the
antiviral effect of IFN-
(Fig. 3) and 10
5 M amrinone was also
ineffective (not shown). Similarly, IBMX (5 × 10
4 M), like milrinone and
amrinone a phosphodiesterase inhibitor, had no effect on virus yield
either in the presence or absence of IFN-
(results not shown).

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Fig. 3.
Effect of T4, milrinone and
amrinone on antiviral action of IFN- in HeLa cells.
T4 or bipyridine was added to
cells either for a 24-h preincubation before a 24-h treatment with
IFN- (1.0 IU/ml) or coincubated for last 4 h of a 24-h IFN-
treatment. T4 potentiated IFN-
action almost 100-fold in both experimental models. Milrinone
potentiated antiviral effect of IFN- but only when applied to cells
in preincubation model. Amrinone was without effect. Significance of
T4 or milrinone potentiation:
* P < 0.05; ** P < 0.01.
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We have previously demonstrated that thyroid hormone potentiation of
IFN-
's antiviral effect in the preincubation experimental model is
inhibited by CHX (11). We show this effect in Fig. 4, along with additional results
demonstrating that the addition of CHX to the cell medium during
treatment with milrinone
(10
6 or
10
5 M) inhibited the
bipyridine's enhancement of IFN's antiviral effect. Thus milrinone's
action requires protein synthesis in a manner similar to the action of
T4 and
T3 in the 24-h preincubation model
(11). The addition of milrinone to a maximally effective concentration
of T4 did not further enhance
IFN-
potentiation (results not shown).

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Fig. 4.
Inhibition by cycloheximide (CHX) of potentiation by
T4 and milrinone of IFN-
antiviral effect. T4 and milrinone
were applied to cells during 24-h preincubation with or without CHX (25 µg/ml). Medium was then removed and replaced with medium containing
IFN- (1.0 IU/ml) for 24 h before antiviral assay.
T4 and milrinone each potentiated
antiviral action of IFN- (* P < 0.05); these effects were completely blocked by CHX.
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Interaction of TETRAC with effects of T4
and milrinone.
We have reported that TETRAC and TRIAC
(10
7 M) do not
potentiate the antiviral action of IFN-
but do, however, block the
protein synthesis-dependent potentiation by
T4 of IFN-
's action (11). We
therefore tested whether TETRAC also blocked milrinone's potentiation in the preincubation experimental model. Results in Fig.
5 indicate that although TETRAC blocked the
antiviral potentiation by T4, there was no effect of TETRAC on the potentiating action of milrinone. Parallel studies using TRIAC and milrinone yielded similar results (not
shown). To further demonstrate the disparate responses to TETRAC of
T4- and milrinone-induced
antiviral potentiation, cells were pretreated for 24 h with
T4, milrinone, and TETRAC before IFN-
treatment. Results in Fig. 5 show that milrinone restored potentiation of IFN-
's antiviral activity in the presence of T4 and TETRAC. These experiments
indicate that the actions of T4
and milrinone in potentiating the effect of IFN-
do not depend on
the identical mechanism.

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Fig. 5.
Interaction of T4, milrinone, and
tetraiodothyroacetic acid (TETRAC) in potentiation of antiviral effect
of IFN- . Cells were pretreated with
T4, milrinone (Mil) or both, with
or without TETRAC, before 24-h IFN- exposure, and effect on virus
yield was then determined. TETRAC blocked potentiation by
T4 but not potentiation by
milrinone. Addition of milrinone to TETRAC and
T4 caused potentiation to be
expressed. *** P < 0.001, significant potentiation by both T4 and milrinone.
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Effect of protein kinase agonists and antagonists.
The effects of PKC agonist, PMA, and PKC inhibitor, CGP-41251, on
potentiation of IFN-
's antiviral effect by
T4 and milrinone are seen in Fig.
6. CGP-41251 has a concentration inhibiting
50% of the maximal response of 5 × 10
8 M for PKC (3); at a
concentration of 5 × 10
10 M, the inhibitor
partially blocked the T4 effect
and completely blocked the milrinone effect. It was not possible to use
higher concentrations of CGP-41251 because of their toxic effect on
subsequent incubation of cells with IFN-
, even though the medium
with inhibitor was replaced with fresh medium before the IFN-
treatment. When used alone before IFN-
but without hormone or
bipyridine, neither CGP-41251 nor PKC agonist PMA had any effect on
virus yield (not shown). In cells treated with either
T4 or milrinone before IFN-
, PMA partially blocked the antiviral potentiation, presumably reflecting partial depletion of PKC in cells. In the presence of
T4 or milrinone and CGP-41251, PMA
reversed the CGP-41251 effect completely in the
T4-treated cells and partially in
the milrinone-treated cells. It is presumed that PMA and CGP-41251 are
mutually inhibitory (22) but permit endogenous diacylglycerol,
generated during exposure of cells to thyroid hormone, to activate PKC,
since the affinity of PKC for diacylglycerol is 250-fold that for
phorbol ester (15).

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Fig. 6.
Effect of CGP-41251 and phorbol 12-myristate 13-acetate (PMA) on
T4 and milrinone potentiation of
antiviral effect of IFN- . CGP-41251 (CGP, 5 × 10 10 M) and PMA
(10 7 M) were added
individually or together during T4
or milrinone preincubations. Medium was then removed and replaced with
fresh medium for IFN- incubation. CGP impaired potentiating effect of both T4 and milrinone
(*** P < 0.001), as did PMA
(not significant with T4;
* P < 0.05 with milrinone).
When added together, effects of CGP and PMA were partially or
completely neutralized. Neither CGP nor PMA altered antiviral effect of
IFN- alone (not shown).
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A similar pattern was seen in studies utilizing the PKA inhibitor
KT-5720 and agonist 8-BrcAMP (Fig. 7).
Partial or complete inhibition of the potentiation by
T4 and milrinone was seen when cells were also pretreated with KT-5720 (5 × 10
7 M). Preincubation with
8-BrcAMP did not affect potentiation of antiviral effect by
T4 or milrinone, but 8-BrcAMP did
reverse the action of KT-5720 in the presence of
T4 and partially reversed the
KT-5720 effect on milrinone potentiation. Neither KT-5720 nor 8-BrcAMP,
in the absence of T4, had any
effect on the subsequent antiviral effect of IFN-
(results not
shown).

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Fig. 7.
Effect of KT-5720 and 8-bromo-cAMP (8-BrcAMP) on
T4 and milrinone potentiation of
antiviral action of IFN- . KT-5720 (KT, 5 × 10 7 M) and 8-BrcAMP (8-Br,
10 3 M) were added
individually or together during T4
or milrinone preincubations. Both
T4 and milrinone enhanced
antiviral action of IFN- significantly
(*** P < 0.001). KT blocked
potentiating effects of T4 and
milrinone (* P < 0.05;
** P < 0.01), whereas 8-Br had
no effect. 8-Br did, however, partially or completely reverse effect of
KT. Neither KT nor 8-Br had any effect on antiviral action of IFN-
alone (not shown).
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We also tested whether the two kinase agonists, PMA and 8-BrcAMP, if
added together to cells for 24 h before IFN-
treatment could
simulate the effects of T4 and
milrinone. Although each agonist alone did not potentiate the IFN-
effect, the two together did enhance the antiviral action (results not
shown), suggesting that T4 and
milrinone exert their effects by stimulating activities of both PKC and
PKA and that both kinase activities are necessary for potentiation.
The effects of the tyrosine kinase inhibitor, genistein (17), on
T4 and milrinone action are shown
in Fig. 8. Genistein was an effective
concentration-dependent inhibitor of
T4 potentiation when both were
added during the 24-h preincubation. On the other hand, genistein had
no effect on the potentiation of the antiviral action of IFN-
by
milrinone. Genistein did not inhibit the antiviral effect of IFN-
alone (not shown), since the inhibitor was washed out before the
IFN-
treatment. Thus tyrosine phosphorylation also plays a role in
potentiation by thyroid hormone, but milrinone's action does not
involve that signaling mechanism.

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Fig. 8.
Inhibition by genistein of potentiation by
T4 but not milrinone of antiviral
action of IFN- . Increasing concentrations of genistein caused
progressive loss of T4
potentiation but no loss of milrinone potentiation. Significant effect
of T4 or milrinone or loss of
potentiation due to genistein:
** P < 0.01;
*** P < 0.001.
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Comparison of ligand binding and transcriptional effects of
T3 and milrinone.
To examine whether milrinone interacts with nuclear TRs, we compared
the ligand binding and transcriptional effects of milrinone with those
of T3. E. coli-expressed TR-
was incubated with
10
9 M
[125I]T3
and increasing concentrations of
T3 or milrinone were added (Fig.
9). A three-fold excess of unlabeled
T3 decreased
[125I]T3
binding by 50%, whereas a 300-fold excess of milrinone had no
significant effect on labeled T3
binding.

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Fig. 9.
Comparison of ligand binding effects of
T3 and milrinone. Tracer
[125I]T3
concentration was 10 9 M. T3 binding to thyroid hormone
receptor- (TR- ) was decreased 50% by a 3-fold excess of
unlabeled T3, whereas a 300-fold
excess of milrinone had no significant effect on hormone binding.
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CV-1 cells, which lack TR (27), were cotransfected with TR-
expression plasmid and a TRE-containing reporter plasmid F2, and the
effects of T3 and milrinone were
examined (Fig. 10). In the absence of
T3, unliganded TR-
repressed
basal transcription as previously reported (2, 26, 28).
T3 in concentrations of
10
6 and
10
8 M stimulated
transcription four- and twofold above basal levels, respectively.
Milrinone (10
6 M) had no
effect on basal repression and did not stimulate transcription. Additionally, 10
6 M
milrinone did not block transcriptional activation by
10
8 M
T3. Similar results were also
observed for these ligands when CV-1 cells were cotransfected with
TR-
expression plasmid and F2 reporter (data not shown).

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Fig. 10.
Comparison of effects of T3 and
milrinone on transcription of TR- . Both
10 6 and
10 8 M
T3 stimulated transcription.
Milrinone (10 6 M) had no
effect on transcription and did not block effect of T3,
10 8 M. Data are shown as
means ± SD.
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Additional studies were performed to examine the effect of TETRAC on
transcriptional activation of TR-
. TETRAC allowed derepression of
basal transcriptional activity and generated weak agonist activity (results not shown), whereas, in additional studies, we have
demonstrated that TRIAC is equipotent with
T3 in functional studies with
TR-
. Our findings suggest that TETRAC can bind to nuclear TR and
function as a weak agonist in contrast to milrinone, which does not
bind to TR. However, it should be noted that, in the potentiation of the antiviral action of IFN-
, neither TETRAC nor TRIAC is an agonist.
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DISCUSSION |
In the present studies, we examined the signal transduction pathways of
a recently recognized action of thyroid hormone in human cells, namely,
the potentiation by T4 and
T3 of the antiviral activity of
homologous IFN-
(11, 13). This is a complex model of hormone action,
in that T4 and
T3 can potentiate the action of
IFN-
by two mechanisms, one that requires protein synthesis and a
second, postnuclear pathway that is independent of protein synthesis
(11). These pathways are depicted in Fig.
11. The first pathway is
susceptible to stimulation by T4,
T3, and
rT3 and is blocked by TETRAC,
whereas the postnuclear pathway is unresponsive to
rT3 and is unaffected by TETRAC
(11). Both pathways are more responsive to
T4 than
T3 in physiological
concentrations, and the effect of
T4 is not altered by coincubation
of cells with PTU. We have demonstrated this potentiation by thyroid
hormone in HeLa and CV-1 cells (H.-Y. Lin, unpublished observations); HeLa cells, like CV-1 cells, are deficient in TR (19, 27).

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Fig. 11.
Proposed protein synthesis-dependent mechanism by which thyroid hormone
potentiates antiviral action of IFN- . In absence of thyroid hormone,
IFN- , via a cell surface receptor, induces phosphorylation of the
signal transduction and activation of transcription protein STAT1 by
tyrosine kinase activity of Janus kinases (JAK1 and JAK2) (4, 8, 18).
Dimerized STAT1 translocates to cell nucleus and binds to an
IFN- -activated sequence element in promoter region of an
IFN- -responsive gene, leading to induction of antiviral proteins
(ER, endoplasmic reticulum). Maximal activity of STAT1 is achieved
when both tyrosine (Tyr) and serine (Ser) residues are phosphorylated
(25), but tyrosine phosphorylation is sufficient to induce antiviral
activity. In present studies, potentiation by thyroid hormone of
IFN- action is shown to require PKC and PKA (serine kinase)
activities as well as tyrosine kinase and to be inhibited by TETRAC. It
is possible that T4 induces tyrosine phosphorylation of STAT1 , but hormone alone is not
antiviral. We postulate that
T4-induced PKC and PKA activities
relate to serine phosphorylation of STAT1 . Inhibition by genistein
of hormone action but not IFN- action implicates tyrosine
phosphorylation in activation of serine kinase(s) relevant to STAT1
rather than direct stimulation of tyrosine phosphorylation of STAT1 .
Milrinone is shown here to require serine kinase but not tyrosine
kinase activity and to effect potentiated antiviral state by a pathway that is not sensitive to TETRAC. A second pathway, postnuclear and CHX
insensitive, by which T4 enhances
antiviral action of IFN- has previously been described (12) and is
also shown here.
|
|
The use of milrinone, which shares structural homologies with thyroid
hormone (14), allowed us to further characterize the interaction of
iodothyronines with the IFN-
signal transduction pathway. The
experiments described here showed that milrinone, at concentrations
that are achieved clinically, also enhanced the antiviral action of
IFN-
by a protein synthesis-dependent mechanism. This action
mimicked that of T4,
T3, and
rT3. Like thyroid hormone,
milrinone had no antiviral activity in the absence of IFN-
.
Milrinone was inactive in the protein synthesis-independent pathway,
and amrinone was wholly inactive in both protein synthesis-dependent and -independent experimental paradigms. The difference in activities of the two bipyridines was not surprising, since X-ray crystallographic analysis has shown that the ring structure of milrinone, but not amrinone, resembles that of iodothyronines (14).
Our observations show that activities of PKC and PKA together are
necessary components of the protein synthesis-dependent potentiation of
IFN-
's action by thyroid hormone and milrinone. PMA and 8-BrcAMP,
when added concurrently but not separately, partially reproduced the
IFN potentiation achieved with thyroid hormone and the bipyridine. That
the roles of PKC and PKA in the protein synthesis-dependent and
-independent pathways are different is shown by the fact that
milrinone, though requiring PKC and PKA in the protein
synthesis-dependent pathway, is inactive in the postnuclear pathway.
Although milrinone and thyroid hormone have structural homologies and
both depend for their action on PKC and PKA in these studies, they do
not enhance the action of IFN-
in the protein synthesis-dependent
pathway by a wholly identical mechanism. That this is the case was
shown by the ability of two deaminated analogues of thyroid hormone,
TETRAC and TRIAC, to block the action of
T4 but not that of milrinone. This
is likely to represent inhibition by TETRAC and TRIAC of binding of
T4 at an extranuclear site that is
linked to kinase activation. We have previously shown that TETRAC
blocks T4 binding to erythrocyte
membranes and also inhibits T4
activation of membrane Ca2+-ATPase
but does not itself increase
Ca2+-ATPase activity (6).
Furthermore, genistein, an inhibitor of tyrosine kinases (17),
inhibited thyroid hormone action but did not affect the potentiating
activity of milrinone. The sites in a signal transduction pathway at
which TETRAC and genistein effect their actions must be
proximal to PKC and PKA activation, or both TETRAC and genistein would
also inhibit milrinone's action. Additional support for this
conclusion was provided by experiments in which a full milrinone
response was achieved in the presence of
T4 with a
T4-inhibiting concentration of
TETRAC.
The apparent dependence of thyroid hormone action on a mechanism that
involves tyrosine kinase activity as well as serine-threonine kinases
(PKC and PKA) suggests that thyroid hormone action is mediated via the
signal transduction pathway that includes activities of the Janus
kinase (JAK) family of tyrosine kinases and proteins involved in signal
transduction and activation of transcription (STAT proteins). This
pathway has been established as a major mechanism for intracellular
signaling initiated by many cytokines including the IFNs (4, 8, 18).
Exposure of the cellular IFN-
receptor to IFN-
results in
receptor tyrosine phosphorylation and activation of JAK1 and JAK2 by
tyrosine transphosphorylation. Subsequently, the 91-kDa protein
STAT1
is also activated by tyrosine phosphorylation, leading to
homodimerization, nuclear translocation of the dimer, and ultimately
binding to the IFN-
response elements on target DNA called IFN-
activation sites. Results of the present studies are consistent with
the hypothesis that one of the actions of thyroid hormone in the HeLa
cell model is stimulation of tyrosine kinase activity in the
JAK-STAT pathway.
We also postulate that the PKC- and PKA-requiring pathway which we have
demonstrated to be shared by the actions of milrinone and thyroid
hormone, distal to steps influenced by deaminated iodothyronines and by
genistein, involves further phosphorylation of STAT1 on serine
residues, resulting in heightened binding affinity of the STAT1-STAT1
dimer for promoter regions on IFN-
-responsive genes. Wen et al. (25)
have shown that both tyrosine and serine phosphorylation are necessary
for maximal activation of transcription by STAT1. We do not believe
that T4 acts via a traditional
nuclear TR in this IFN-
-HeLa cell model but, rather, that IFN-
potentiation is achieved by a thyroid hormone-directed protein kinase
cascade, which is summarized in Fig. 11.
Although these studies were carried out in a cell line deficient in TR,
we nonetheless pursued the possibility that milrinone and TETRAC were
capable of interacting with TR. The lack of interaction of TETRAC with
TR-
is consistent with the action of TETRAC in the antiviral studies
at an extranuclear binding site for thyroid hormone. We studied the
possibility that milrinone could bind to nuclear receptors for thyroid
hormone and that such receptors were associated with potentiation of
the cellular antiviral response directed by IFN-
. Milrinone,
however, was found not to compete with
T3 for binding sites on
bacterially expressed TR-
. In addition, milrinone did not
transactivate or block T3-mediated
transcriptional activity measured via the TRE F2 that was cotransfected
with TR-
or TR-
. These results indicate that milrinone
1) does not have sufficient
structural homology with thyroid hormone to permit binding of the
bipyridine to a principal nuclear receptor for T3 and
2) does not potentiate the antiviral
activity of IFN-
by means of a mechanism dependent on TR-
or
TR-
.
 |
ACKNOWLEDGEMENTS |
We thank Jeannie Whang and Dr. Ying Liu for valuable technical
assistance in ligand binding and cotransfection studies.
 |
FOOTNOTES |
This work was supported in part by 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.
Address for reprint requests: P. J. Davis, Dept. of Medicine A-57,
Albany Medical College, Albany, NY 12208.
Received 11 March 1997; accepted in final form 21 May 1997.
 |
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