| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
-Induced HLA-DR Expression1
,,,
*
Division of Molecular and Cellular Medicine, Department of Medicine, and
Department of Biochemistry, Albany Medical College, Albany, NY 12208; and
Veterans Affairs Healthcare Network Upstate New York, Albany, NY 12208
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-induced HLA-DR expression. IFN--induced HLA-DR
expression requires activation of STAT1
and induction of the Class
II trans-activator, CIITA. HeLa and CV-1 cells treated only with
L-thyroxine (T4) demonstrated increased
tyrosine phosphorylation and nuclear translocation (= activation) of
STAT1; this hormone effect on signal transduction, and
T4 potentiation of IFN--induced HLA-DR expression, were
blocked by the inhibitors CGP 41251 (PKC) and genistein (tyrosine
kinase). Treatment of cells with T4-agarose also caused
activation of STAT1. In the presence of IFN-, T4
enhanced cytokine-induced STAT1 activation. Potentiation by
T4 of IFN- action was associated with increased mRNA for
both CIITA and HLA-DR, with peak enhancement at 16 h (CIITA), and
2 d (HLA-DR). T4 increased IFN--induced HLA-DR
protein 2.2-fold and HLA-DR mRNA fourfold after 2 d. Treatment
with actinomycin D after induction of HLA-DR mRNA with IFN-, with or
without T4, showed that thyroid hormone decreased the
t1/2 of mRNA from 2.4 to 1.1 h. HeLa and
CV-1 cells lack functional nuclear thyroid hormone receptor.
Tetraiodothyroacetic acid (tetrac) and 3,5,3'-triiodo-thyroacetic acid
(triac) blocked T4 potentiation of IFN--induced HLA-DR
expression and T4 activation of STAT1. These studies
define an early hormone recognition step at the cell surface that is
novel, distinct from nuclear thyroid hormone receptor, and blocked by
tetrac and triac. Thus, thyroid hormone potentiation of IFN--induced
HLA-DR transcription is mediated by a cell membrane hormone binding
site, enhanced activation of STAT1, and increased CIITA induction. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
in several other cell
types (3, 4, 5). We have shown that the induction of HLA-DR by IFN-
in HeLa cells is potentiated by L-thyroxine
(T4)3 through a
mechanism that is protein kinase A (PKA)- and protein kinase C
(PKC)-dependent (6). In this report, we describe the involvement of
STAT1 and the Class II trans activator protein, CIITA, known to be
an essential component of the IFN--generated HLA-DR induction
process (7, 8, 9), in hormone potentiation of IFN--induced HLA-DR
expression. Thyroid hormone is shown to stimulate tyrosine
phosphorylation and nuclear translocation of STAT1. The mechanism of
hormone action in potentiation of IFN--associated HLA-DR induction
is thought to be primarily at the level of kinase activation of
STAT1. |
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
HeLa cells were obtained from American Type Culture Collection (ATCC, Manassas, VA), and CV-1 cells were generously provided by Dr. Paul M. Yen (National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health, Bethesda, MD). Cells were grown in DMEM supplemented with 10% FBS, as described previously (10). Thyroid hormone was removed from the serum by ion exchange resin, according to the method of Samuels et al. (11). In 10% hormone-depleted serum-supplemented medium (SSM) used in the HLA-DR experiments, the total and free concentrations of T4 were 0.9 x 10-9 and 0.4 x 10-12 M, respectively. The total 3,5,3'-L-triiodothyronine (T3) concentration was <10-10 M (10), and the free T3 concentration was undetectable (12). In the signal transduction experiments, cells were maintained in 0.25% hormone-depleted SSM, containing total and free T4 concentrations of 2.3 x 10-11 M and 10-14 M, respectively, and a total T3 concentration of <2.5 x 10-11 M.
Reagents
Recombinant human IFN- was obtained from BioSource
International (Camarillo, CA). L-T4,
L-T3, D-T4,
D-T3, 3,3',5'-triiodothyronine (reverse
T3), 3,5-diiodothyronine (T2),
tetraiodothyroacetic acid (tetrac), 3,5,3'-triiodothyroacetic acid
(triac), actinomycin D, T4-agarose, and protein A-agarose
were purchased from Sigma (St. Louis, MO). Stock hormone and analogue
solutions were prepared in 0.04 N KOH, 4% (v/v) propylene glycol at a
concentration of 10-4 M, and diluted in culture medium.
Mouse anti-human HLA-DR -chain-specific Ab,
peroxidase-conjugated rabbit anti-mouse IgG, and goat
anti-rabbit IgG were purchased from Dako Corporation (Carpinteria,
CA). Anti-phosphotyrosine-agarose and polyclonal STAT1 Ab were
obtained from Transduction Laboratories (Lexington, KY), and another
polyclonal STAT1 Ab from Upstate Biotechnology (UBI, Lake Placid,
NY). The chemiluminescence detection system (ECL kit) was purchased
from Amersham (Arlington Heights, IL). CGP 41251, a PKC inhibitor, was
a gift from Ciba-Geigy (Basel, Switzerland), and genistein, a protein
tyrosine kinase (PTK) inhibitor, was obtained from ICN Biochemicals
(Costa Mesa, CA). Stock solutions of the two inhibitors were prepared
in DMSO, and dilutions used in cell treatments contained 
1%
DMSO.
Northern blotting of HLA-DR and CIITA mRNA
Confluent HeLa cells grown in 25-cm2 tissue
flasks were treated with 10% thyroid hormone-depleted SSM overnight.
Either 10-7 M T4, 100 IU/ml IFN-, or
IFN- plus T4 were added to cells for 1 to 3 d in
HLA-DR mRNA studies, and for 6 to 24 h in CIITA studies. Total
cellular RNA was isolated utilizing a single step RNA procedure
(Ultra-spec, Biotecx Laboratories, Houston, TX). For Northern blotting,
10 µg RNA/well were electrophoretically separated on an agarose gel
and transferred to MagnaCharge nylon membranes (MSI, Westborough, MA)
by the pressure transfer method. An HLA-DR plasmid purchased from ATCC
(Cat. No. 57393) contained a cDNA insert of 1.3 Kb specific for HLA-DR
-chain. A plasmid containing the cDNA for the Class II trans
activator, CIITA (9), was kindly provided by Dr. C.-H. Chang of Yale
University (New Haven, CT). A 1.8-kb fragment of the cDNA was used for
Northern blotting. The HLA-DR- and CIITA probes were labeled with
[32P]dCTP by random-primed synthesis (13), and
hybridization procedures followed those outlined by Maniatis et al.
(14). The filters were exposed to Kodak AR film, and the band
intensities were measured by digital imaging. To correct for variation
in total RNA between samples, probes for either
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or 28S ribosomal RNA
were utilized, and the band intensities for HLA-DR and CIITA were
normalized appropriately. The effect of inhibition of transcription on
levels of HLA-DR and CIITA mRNA was studied with actinomycin D (5
µg/ml) (15) in cell incubations lasting 30 min to 3 h.
Effect of thyroid hormone on the expression of HLA-DR induced by
IFN-
Confluent HeLa cells grown in six-well trays were treated with
fresh 10% thyroid hormone-depleted SSM for 24 h. In the studies
with thyroid hormone analogues, IFN- (100 IU/ml) was added with or
without 10-7 M L-T4,
D-T4, L-T3,
D-T3, reverse T3, T2,
tetrac, or triac, and cell cultures were incubated for 2 d. Cells
were then harvested, washed twice with PBS (PBS, containing 137 mM
NaCl, 2.7 mM KCl, 4.3 mM
Na2HPO4·7H2O, and 1.4 mM
KH2PO4), resuspended in PBS with 0.1% Nonidet
P-40, and sonicated for 10 s. Protein concentrations were
determined by Bio-Rad protein assay (Bio-Rad, Hercules, CA).
Immunoblotting of HLA-DR
Discontinuous SDS-polyacrylamide gel electrophoresis was
performed using 12% acrylamide under reducing conditions. Twenty
micrograms protein from each sample were applied. Proteins were then
transferred to Immobilon membranes (Millipore, Bedford, MA) by
electroblotting. After blocking with 5% milk in Tris-buffered saline
(TBS, 100 mM Tris · HCl, pH 7.5, 0.9% NaCl) containing 0.1%
Tween, membranes were incubated for 2 h at room temperature with
mouse anti-human HLA-DR -chain-specific Ab. The second Ab was
peroxidase-conjugated rabbit anti-mouse IgG. Immunoblots were
visualized by chemiluminescence and quantitated by digital imaging
(BioImage, Millipore). The intensity of the 34-kDa bands was expressed
as the integrated OD (IOD), which is a function of the band intensity
multiplied by its area. In all experiments, except where indicated in
the figure legends, the IOD in the sample treated with IFN-, 100
IU/ml, was normalized to 100%, and the other sample IODs were
expressed as a percent of that sample within the same experiment.
Graphic results shown are the means ± SE of two or more
experiments, and the blots shown are from representative
experiments.
Preparation of nuclear fractions
Confluent HeLa or CV-1 cells grown in 100-mm culture dishes were
treated with 0.25% hormone-depleted SSM for 48 h. Hormone or
analogues and IFN-, with or without kinase inhibitor, were then
added at different time points as indicated. Cells were harvested, and
nuclear extracts were prepared as follows: cell cultures were washed
twice with ice-cold PBS and lysed in hypotonic buffer (20 mM HEPES, pH
7.9, 10 mM KCl, 0.1 mM Na3VO4, 1 mM EDTA,
10% glycerol, 1 mM PMSF, 3 µg/ml aprotinin, 1 mg/ml pepstatin, 20 mM
NaF, and 1 mM DTT) with 0.2% Nonidet P-40 on ice for 10 min. After
centrifugation at 4°C and 13,000 rpm for 1 min, supernatants were
collected as cytoplasmic extracts. Nuclear extracts were prepared
according to the method of Wen et al. (16) by resuspension of the crude
nuclei in high salt buffer (hypotonic buffer with 20% glycerol and 420
mM NaCl) at 4°C with rocking for 30 min. The supernatants were
collected after centrifugation at 4°C and 13,000 rpm for 10
min.
Phosphotyrosine immunoprecipitation and immunoblotting of STAT1
Following normalization of protein content, immunoprecipitation
was performed using anti-phosphotyrosine-agarose (PY-20 agarose).
After overnight incubation at 4°C, the agarose was washed three times
with the recommended buffer (Transduction Laboratories) containing
0.2% Nonidet P-40, and the immunoprecipitates were eluted and
separated by discontinuous SDS-polyacrylamide gel electrophoresis.
Proteins were transferred to Immobilon membranes (Millipore) by
electroblotting. After blocking with 5% milk in Tris-buffered saline
containing 0.1% Tween, membranes were incubated with 1:1000 polyclonal
rabbit anti-mouse STAT1 Ab overnight. The secondary Ab was goat
anti-rabbit IgG (1:1000). Immunoblots were visualized by
chemiluminescence and quantitated by digital imaging.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
on tyrosine phosphorylation and nuclear translocation of
STAT1
The effects of thyroid hormone on tyrosine phosphorylation and
nuclear translocation of STAT1, in the presence or absence of a PKC
or tyrosine kinase inhibitor, tetrac, triac or IFN-, were studied.
Hormone, analogues or T4-agarose were added to medium
for the times indicated. Inhibitors or solvent were applied to cells
for 70 min. Cells were then harvested, and nuclear fractions from each
sample were prepared. Proteins from these fractions were
immunoprecipitated with anti-phosphotyrosine Ab, and proteins from
the immunoprecipitates were electrophoresed and immunoblotted with
STAT1 Ab. In Figure 1
A,
increased accumulation of nuclear tyrosine-phosphorylated STAT1 in
the presence of T4, 10-7 M, is seen in HeLa
cell extracts. This accumulation was inhibited by the PKC-, -ßI,
-ßII, and - inhibitor, CGP 41251 (0.1 nM) and by the tyrosine
kinase inhibitor, genistein (50 µg/ml). IFN- was not present in
the incubations from which these samples were derived.
|
. The blot shown in Figure 1
B again demonstrates tyrosine phosphorylation and nuclear
accumulation (activation) of STAT1 in the presence of
T4. Tetrac and triac, 10-7 M, did not cause
tyrosine phosphorylation of STAT1, but in concentrations of
10-7 to 10-5 M did block the effect of
T4 on this trans activator. A physiologic concentration of
T3 (10-10 M) did not activate STAT1, and
10-7 M T3 was not as effective as
10-7 M T4 (results not shown). Results of
studies with T4-agarose are shown in Figure 1C.
CV-1 cells treated with 10-7 M T4 for 30 min
showed activation of STAT1 in nuclear fractions (lane
3), whereas no activation was seen in the control lane
(lane 1) or in cells exposed to protein
A-agarose (lane 2). T4-agarose
containing 10-7 M T4, whether washed
(lane 5) or not (lane
4), caused STAT1 activation as seen with T4
alone. In Figure 1D the results of HeLa cell treatment for
30 min with T4, 10-7 M, and/or IFN-, 1
IU/ml, are shown. There is an additive effect of T4 and the
cytokine on the activation of STAT1.
Time course of the appearance of CIITA mRNA in response to IFN-
and thyroid hormone
Samples of total RNA from cells treated with T4,
IFN-, or both for 6, 16, and 24 h were prepared and analyzed
for CIITA, HLA-DR, or GAPDH mRNA by Northern blotting. As shown in
Figure 2, CIITA mRNA appeared in 6 h
in samples from cells treated with IFN-, and levels of this mRNA
were enhanced in cells treated with T4 in addition to
IFN-; in the 6- and 16-h samples, T4 enhanced the
IFN- effect twofold. The appearance of CIITA mRNA in 6 h
preceded the appearance of HLA-DR mRNA in 16 h (Fig. 2). There was
no CIITA mRNA induced in cells treated for 24 h with
T4, alone. With inhibition of transcription by
actinomycin D, 5 µg/ml, CIITA mRNA disappeared equally quickly in
IFN--treated cells in the presence or absence of T4
(results not shown).
|
-induced
HLA-DR mRNA
The potentiation by T4 of IFN--induced HLA-DR
expression is consistent with enhancement by T4 of either
the transcription of the HLA-DR gene or translation of its mRNA. As
shown in Figure 3, the abundance of
HLA-DR mRNA in HeLa cells treated with T4 and IFN- was
increased 1.8- and fourfold after 1 and 2 d, respectively,
compared with cells treated with IFN- alone. The abundance of HLA-DR
mRNA dropped sharply by 3 d in the cells treated with both hormone
and IFN-, compared with cells treated with IFN- alone. To
evaluate the possible role of T4 in the stabilization of
HLA-DR mRNA, actinomycin D (5 µg/ml) was added to cell cultures for 1
to 3 h after a 1-d incubation with IFN-, with or without
T4. Results are shown in Figure 4 and indicate that thyroid hormone
accelerated the rate of disappearance of HLA-DR mRNA. With IFN-
alone, the t1/2 of the mRNA was 2.4 h, and
with the addition of T4 the t1/2 was
1.1 h.
|
|
-induced
HLA-DR expression
To relate the accumulation of HLA-DR to appearance of its message,
the time course of hormone potentiation was studied. The expression of
HLA-DR induced by 100 IU/ml IFN- increased most sharply in 2 to
3 d (Fig. 5). When
T4, 10-7 M, was added simultaneously with
100 IU/ml IFN- to cells, the potentiation of IFN-’s action
reached a maximum at day 2, a 2.2-fold increase, followed by a
progressive loss of hormone potentiation from 3 to 5 d. Detectable
HLA-DR Ag accumulated in cells incubated with T4, alone,
for 5 d, to a level of 10 ± 4% of the amount found in
cultures treated with IFN-, alone, for 2 d (Fig. 5).
|
-induced HLA-DR expression
In cells treated with T4 and IFN-, 100 IU/ml,
there was potentiation of IFN--induced HLA-DR expression by the
hormone, as shown previously (6), and a 56% reduction in that
potentiation caused by genistein in a very low concentration of 0.05
µg/ml, shown in Figure 6A.
In contrast, the same concentration of inhibitor caused no reduction in
IFN--induced HLA-DR expression in cells treated without
T4 (Fig. 6B). Even with the differences
in concentrations and incubation conditions between these studies of
HLA-DR expression and those of STAT1 activation illustrated in
Figure 1A (genistein 0.05 µg/ml for 2 d, Fig. 6, compared with 50 µg/ml for 70 min, Fig. 1A), there
is indication that in each experimental model the effect of
T4 is blocked by this tyrosine kinase inhibitor.
|
-induced HLA-DR
expression in HeLa cells
The effects of 10-7 M
L-T4, L-T3,
D-T4, D-T3, reverse
T3, and T2 on potentiation of HLA-DR expression
induced in 2 d by IFN- (100 IU/ml) were examined and are shown
in Figure 7. L-T4
and L-T3 enhanced IFN--induced HLA-DR levels
1.7- and 1.8-fold, respectively, in four experiments. In contrast,
D-T4, D-T3, reverse
T3, and T2 did not enhance the expression of
HLA-DR induced by IFN-.
|
for 2 d, did not potentiate IFN--induced HLA-DR
expression (Fig. 8). However, both
analogues did block T4 potentiation of the IFN- effect
(Fig. 8), as did D-T4 (results not shown). That
tetrac and triac block T4 potentiation of IFN-
correlates well with the ability of these two analogues to block
T4-enhanced tyrosine phosphorylation and nuclear
translocation of STAT1, as presented in Figure 1B. Since
HeLa cells do not contain functional thyroid hormone receptor (TR)
(17), this cannot be a nucleus-mediated effect.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
in the absence of
thyroid hormone requires activation of the Janus kinase/STAT (JAK-STAT)
pathway (18). This pathway includes multiple sites of tyrosine
phosphorylation. These sites are on the IFN- receptor, on Janus
kinases 1 and 2, and on STAT1, leading to nuclear translocation of a
STAT1 homodimer (19). Maximal transcriptional activity of
tyrosine-phosphorylated STAT1 is obtained when phosphorylation of
serine 727 also occurs (16). The latter has been thought to be
accomplished by mitogen-activated protein kinase (MAPK) (16), but
recent evidence suggests another serine kinase may be involved (20).
The MAPK pathway is dependent upon activities of PKC and PKA (21, 22),
kinases that we have previously shown to be essential for thyroid
hormone to potentiate IFN--induced HLA- DR expression (6). In the
present studies we considered several mechanisms by which physiologic
concentrations of thyroid hormone may potentiate this action of
IFN-. These mechanisms included activation of STAT1 and induction
of CIITA expression, transcription of HLA-DR mRNA and
increased efficiency of translation of the mRNA.
Because there is little or no functional TR in HeLa cells (17) or CV-1
cells (12), the first step in the potentiating effect of thyroid
hormone is not the binding of T4 or T3 to
TR. The evidence presented here supports instead a mechanism of thyroid
hormone action involving PKC and PTK in the STAT signal transduction
pathway. We found that thyroid hormone induces tyrosine phosphorylation
and nuclear translocation of STAT1 in HeLa and CV-1 cells in the
absence of IFN-. This effect is blocked by CGP 41251, an inhibitor
of PKC activity (23), and by the PTK inhibitor, genistein. In
additional studies with T4 and IFN-, the hormone
potentiated cytokine-induced tyrosine phosphorylation and nuclear
translocation of STAT1 in cells treated simultaneously with
hormone and IFN-. As noted above, serine phosphorylation of
tyrosine-phosphorylated STAT1 promotes maximal transcriptional
activation by this protein (16). Thus, we propose that thyroid hormone
enhances IFN--induced phosphorylation and nuclear translocation of
STAT1, leading to hormone-potentiated expression of CIITA and HLA-DR
genes. The apparent participation of PKC activity in this mechanism is
consistent with our previously reported finding of roles for this
signal-transducing kinase family in hormone-induced potentiation of
IFN- action on HLA-DR expression (6).
Benveniste et al. (24) and Lee et al. (25) have previously shown that
IFN--induced Class II Ag expression is PKC and PTK-dependent. Their
studies involved somewhat high concentrations (up to 100 µM) of the
PKC inhibitors. We have previously shown that two relatively specific
inhibitors of PKC, staurosporine and CGP 41251, at nanomolar
concentrations, enhance IFN--induced HLA-DR expression and
staurosporine inhibits such expression at submicromolar concentrations
(6). Important to note, however, is that nanomolar staurosporine and
CGP 41251 levels inhibited thyroid hormone’s potentiation of IFN-
action in this model (6). Thus, very low concentrations of these
agents allow us to distinguish between PKC participation in the IFN-
effect, alone, and in the action of iodothyronine on
IFN--induced HLA-DR expression. Further, we have shown in the
present study that a very low concentration of genistein (0.05 µg/ml)
distinguishes between the action of IFN- and the potentiating effect
of thyroid hormone. Thyroid hormone potentiation of IFN--induced
HLA-DR expression was inhibited by genistein, 0.05 µg/ml, whereas
concentrations of 5 to 50 µg genistein/ml were required by us and
others (25) to block the action of IFN-, alone, on Class II Ag
expression. This suggests that the tyrosine kinase activities affected
by genistein are different in the cases of T4 potentiation
and of IFN- action.
We confirmed that IFN- increased the abundance of CIITA mRNA and
showed that thyroid hormone, in cells exposed to both IFN- and the
hormone, further enhanced steady state levels of CIITA mRNA after as
little as 6 h of treatment. Thyroid hormone, alone, did not induce
increased levels of CIITA mRNA, nor did the hormone affect the
disappearance rate of CIITA mRNA. Thus, potentiation by iodothyronine
of the effect of IFN- on HLA-DR expression appears to be mediated by
increased abundance of CIITA mRNA. This precedes the
appearance of increased steady state levels of HLA-DR mRNA that are
also caused by thyroid hormone, relative to those induced by IFN-,
alone.
Stabilization of HLA-DR mRNA did not contribute to the thyroid hormone
effect. When actinomycin D was added to HeLa cells after induction of
HLA-DR with IFN-, T4 was shown to shorten the
t
of HLA-DR mRNA, rather than increase it. The increased
expression of HLA-DR Ag caused by thyroid hormone is therefore the net
result of increased abundance of mRNA tempered by a shortened
t of the mRNA. The increased ratio of steady state
abundance of HLA-DR mRNA (fourfold increase) compared with that of
HLA-DR protein (twofold increase after potentiation of IFN- action
by T4 for 2 d) does not support an action of the
hormone to increase efficiency of translation of HLA-DR mRNA. Rather,
the decreased t of HLA-DR mRNA in the presence of
T4 probably accounts for this difference.
Preceding kinase activation by iodothyronines in the IFN--MHC class
II model is interaction of thyroid hormone with a cell surface binding
site whose structure-activity characteristics are described, but whose
precise link to kinase-mediated signal transduction is incompletely
known. The binding site recognizes physiologic concentrations of
T4 and T3 (10-7 M and
10-10 M, respectively), but is not activated by
D-analogues of thyroid hormone, reverse T3 or
deaminated hormone analogues (Figs. 1, 7, 8). However, while tetrac,
triac, and D-T4 were not agonists, they did
block the potentiation by T4 of IFN- action in the
HLA-DR model (Fig. 8; D-T4 results not shown), and both
tetrac and triac blocked T4-induced tyrosine
phosphorylation and nuclear accumulation of STAT1 (Fig. 1B). We have previously shown that tetrac also blocks
T4 potentiation of the antiviral action of IFN- (12).
Reverse T3 had neither an agonist nor antagonist effect on
IFN--induced HLA-DR expression. These structure-activity
relationships of hormone analogues are not consistent with prior
studies of analogue binding to TR (26, 27, 28), and, as pointed out above,
TR is functionally absent from HeLa and CV-1 cells (12, 17).
The nature of the initial step in potentiation by thyroid hormone of
the action of IFN- on HLA-DR expression is not yet clear. That
agarose-T4 was as effective as T4 in the
present studies indicates that a cell surface binding site for the
hormone is involved in hormone action. Consistent with the initiation
of hormone action at the plasma membrane is recent finding that
monodansylcadaverine, an inhibitor of the endocytotic pathway of
thyroid hormone uptake (29), does not affect the action of
T4 on phosphorylation of STAT1 (unpublished
observations). We have previously described in human cells
(erythrocytes) the existence of high affinity plasma membrane binding
sites for thyroid hormone that bind T4, T3 (30, 31), and tetrac (32). Such structure-activity relationships of thyroid
hormone are similar to those reported in the present HLA-DR studies,
but we have not yet determined that comparable sites exist on HeLa or
CV-1 cells.
The mechanism of action of thyroid hormone in the IFN-/HLA-DR model
studied here thus includes interaction with a novel plasma membrane
hormone-recognition site, PTK and PKC activation, STAT1 tyrosine
phosphorylation and nuclear translocation, CIITA gene expression, and,
subsequently, HLA-DR expression. There is a small effect of
iodothyronines on tyrosine phosphorylation of STAT1 in the absence
of IFN-, and this may underlie the ability of the hormone, alone, to
induce very limited HLA-DR expression (see Fig. 5). Thyroid hormone can
also activate MAPK in the absence of IFN- (33). As noted earlier,
however, T4 in the absence of IFN- achieves no
substantive increase in steady state abundance of mRNAs for HLA-DR and
CIITA (Figs. 2 and 3). Indeed, T4 increases the
turnover of HLA-DR mRNA 2.2-fold, a factor that limits the magnitude of
hormone potentiation of IFN--induced HLA-DR expression. The action
of IFN- on the IFN- receptor and on tyrosine phosphorylation of
STAT1 are presumed to be critical steps in transduction of the IFN
signal and must take place to obtain potentiation by thyroid hormone of
the cytokine’s action.
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Paul J. Davis, Department of Medicine A-57, Albany Medical College, Albany, NY 12208. E-mail address:
3 Abbreviations used in this paper: T4, L-thyroxine; CIITA, Class II trans activator; MAPK, mitogen-activated protein kinase; PKC, protein kinase C; PTK, protein tyrosine kinase; T3, 3,5,3'-L-triiodothyronine; reverse T3, 3,3',5'-triiodothyronine; T2, 3,5-diiodothyronine; tetrac, tetraiodothyroacetic acid; triac, 3,5,3'-triiodothyroacetic acid; SSM, serum-supplemented medium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IOD, integrated OD; TR, nuclear thyroid hormone receptor.
Received for publication January 9, 1998. Accepted for publication March 23, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and interleukin 6 decrease nuclear thyroid hormone receptor capacity in a liver cell line. Eur. J. Endocrinol. 131:307., TNF-, and TGF-ß. J. Immunol. 154:2846.[Abstract]
-induced HLA-DR expression is protein kinase A- and C-dependent. J. Interferon Cytokine Res. 16:17.[Medline]
by interferon- requires a trans-acting protein. Proc. Natl. Acad. Sci. USA 85:4672. mediated by the transactivator CIITA. Science 265:106. in cultured human cells. J. Clin. Endocrinol. Metab. 79:62.[Abstract]
. Am. J. Physiol. 273:C1225. expression is involved in IFN- induction of the class II transactivator and class II MHC genes. J. Immunol. 157:1559.[Abstract]
activates RAF-1 by direct phosphorylation. Nature 364:249.[Medline]
-induced astrocyte class II major histocompatibility complex gene expression is associated with both protein kinase C activation and Na+ entry. J. Biol. Chem. 266:18119.-induced expression of the class II MHC gene. Am. J. Physiol. 268:C127.- and ß- forms for the acetic acid analog and failure of the human testis and kidney alpha-2 products to bind T3. Mol. Endocrinol. 4:227.This article has been cited by other articles:
|
|
 |
|
  H.-Y. Lin, M. Sun, H.-Y. Tang, C. Lin, M. K. Luidens, S. A. Mousa, S. Incerpi, G. L. Drusano, F. B. Davis, and P. J. Davis L-Thyroxine vs. 3,5,3'-triiodo-L-thyronine and cell proliferation: activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase Am J Physiol Cell Physiol, May 1, 2009; 296(5): C980 - C991. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Xu, K. Ravid, and B. D. Smith Major Histocompatibility Class II Transactivator Expression in Smooth Muscle Cells from A2b Adenosine Receptor Knock-out Mice: CROSS-TALK BETWEEN THE ADENOSINE AND INTERFERON-{gamma} SIGNALING J. Biol. Chem., May 23, 2008; 283(21): 14213 - 14220. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Shih, S. Zhang, H. J. Cao, S. Boswell, Y.-H. Wu, H.-Y. Tang, M. R. Lennartz, F. B. Davis, P. J. Davis, and H.-Y. Lin Inhibitory effect of epidermal growth factor on resveratrol-induced apoptosis in prostate cancer cells is mediated by protein kinase C-{alpha} Mol. Cancer Ther., November 1, 2004; 3(11): 1355 - 1364. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H.-Y. Tang, H.-Y. Lin, S. Zhang, F. B. Davis, and P. J. Davis Thyroid Hormone Causes Mitogen-Activated Protein Kinase-Dependent Phosphorylation of the Nuclear Estrogen Receptor Endocrinology, July 1, 2004; 145(7): 3265 - 3272. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Shih, S. Zhang, H. J. Cao, H.-Y. Tang, F. B. Davis, P. J. Davis, and H.-Y. Lin Disparate Effects of Thyroid Hormone on Actions of Epidermal Growth Factor and Transforming Growth Factor-{alpha} Are Mediated by 3',5'-Cyclic Adenosine 5'-Monophosphate-Dependent Protein Kinase II Endocrinology, April 1, 2004; 145(4): 1708 - 1717. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Higashi, Y. Inagaki, N. Suzuki, S. Mitsui, A. Mauviel, H. Kaneko, and I. Nakatsuka Y-box-binding Protein YB-1 Mediates Transcriptional Repression of Human alpha 2(I) Collagen Gene Expression by Interferon-gamma J. Biol. Chem., February 7, 2003; 278(7): 5156 - 5162. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Shih, F. B. Davis, H.-Y. Lin, and P. J. Davis Resveratrol Induces Apoptosis in Thyroid Cancer Cell Lines via a MAPK- and p53-Dependent Mechanism J. Clin. Endocrinol. Metab., March 1, 2002; 87(3): 1223 - 1232. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Pappa, J. M M Lawson, V. Calder, P. Fells, and S. Lightman T cells and fibroblasts in affected extraocular muscles in early and late thyroid associated ophthalmopathy Br. J. Ophthalmol., May 1, 2000; 84(5): 517 - 522. [Abstract] [Full Text]
|
|
|
|
|
|
A. Miyazaki, H. Shimura, T. Endo, K. Haraguchi, and T. Onaya Tumor Necrosis Factor-{alpha} and Interferon-{gamma} Suppress Both Gene Expression and Deoxyribonucleic Acid-Binding of TTF-2 in FRTL-5 Cells Endocrinology, September 1, 1999; 140(9): 4214 - 4220. [Abstract] [Full Text]
|
|
|
|
|
|
G. A. Brent Thyroid hormone action: down novel paths Focus on "Thyroid hormone induces activation of mitogen-activated protein kinase in cultured cells" Am J Physiol Cell Physiol, May 1, 1999; 276(5): C1012 - C1013. [Full Text] [PDF]
|
|
|
|
|
|
H.-Y. Lin, F. B. Davis, J. K. Gordinier, L. J. Martino, and P. J. Davis Thyroid hormone induces activation of mitogen-activated protein kinase in cultured cells Am J Physiol Cell Physiol, May 1, 1999; 276(5): C1014 - C1024. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. J. Davis, A. Shih, H.-Y. Lin, L. J. Martino, and F. B. Davis Thyroxine Promotes Association of Mitogen-activated Protein Kinase and Nuclear Thyroid Hormone Receptor (TR) and Causes Serine Phosphorylation of TR J. Biol. Chem., November 22, 2000; 275(48): 38032 - 38039. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
