Transforming Growth Factor-ß1 Down-Regulation of Major Histocompatibility Complex Class I in Thyrocytes: Coordinate Regulation Of Two Separate Elements by Thyroid-Specific as Well as Ubiquitous Transcription Factors
Giorgio Napolitano,
Valeria Montani,
Cesidio Giuliani,
Simonetta Di Vincenzo,
Ines Bucci,
Valentina Todisco,
Giovanna Laglia,
Anna Coppa,
Dinah S. Singer,
Minoru Nakazato,
Leonard D. Kohn,
Giulia Colletta and
Fabrizio Monaco
Chair of Endocrinology (G.N., V.M., C.G., S.D.V., I.B., V.T., G.L.,
F.M.) Department of Medicine and Department of Oncology and
Neuroscience (G.C.) University "G. DAnnunzio", Chieti,
Italy
66100
Department of Experimental Medicine and Pathology
(A.C.) University "La Spienza", Rome, Italy 00161
Experimental Immunology Branch (D.S.S.) National Cancer
Institute and Metabolic Diseases Branch (M.N., L.D.K.) National
Institute of Diabetes and Digestive and Kidney Diseases National
Institutes of Health, Bethesda, Maryland 20892-1800
 |
ABSTRACT
|
---|
Transforming growth factor (TGF)-ß1-decreased
major histocompatibility complex (MHC) class I gene expression in
thyrocytes is transcriptional; it involves trans factors
and cis elements important for hormone- as well as
iodide-regulated thyroid growth and function. Thus, in rat FRTL-5
thyrocytes, TGF-ß1 regulates two elements within -203 bp of the
transcription start site of the MHC class I 5'-flanking region:
Enhancer A, -180 to -170 bp, and a downstream regulatory element
(DRE), -127 to -90 bp, that contains a cAMP response element
(CRE)-like sequence. TGF-ß1 reduces the interaction of a NF-
B
p50/fra-2 heterodimer (MOD-1) with Enhancer A while increasing its
interaction with a NF-
B p50/p65 heterodimer. Both reduced MOD-1 and
increased p50/p65 suppresses class I expression. Decreased MOD-1 and
increased p50/p65 have been separately associated with the ability of
autoregulatory (high) concentrations of iodide to suppress thyrocyte
growth and function, as well as MHC class I expression. TGF-ß1 has
two effects on the downstream regulatory element (DRE). It increases
DRE binding of a ubiquitously expressed Y-box protein, termed TSEP-1
(TSHR suppressor element binding protein-1) in rat thyroid cells;
TSEP-1 has been shown separately to be an important suppressor of the
TSH receptor (TSHR) in addition to MHC class I and class II expression.
It also decreases the binding of a thyroid-specific trans
factor, thyroid transcription factor-1 (TTF-1), to the DRE, reflecting
the ability of TGF-ß1 to decrease TTF-1 RNA levels.
TGF-ß1-decreased TTF-1 expression accounts in part for
TGF-ß1-decreased thyroid growth and function, since decreased
TTF-1 has been shown to decrease thyroglobulin, thyroperoxidase, sodium
iodide symporter, and TSHR gene expression, coincident with decreased
MHC class I. Finally, we show that TGF-ß1 increases c-jun
RNA levels and induces the formation of new complexes involving
c-jun, fra-2, ATF-1, and c-fos, which react
with Enhancer A and the DRE. TGF-ß1 effects on c-jun may
be a pivotal fulcrum in the hitherto unrecognized coordinate regulation
of Enhancer A and the DRE.
 |
INTRODUCTION
|
---|
Transforming growth factor-ß (TGF-ß) polypeptides regulate the
growth, function, and immune properties of cells, decreasing, for
example, major histocompatibility complex (MHC) class I and class II
expression basally or after
-interferon (IFN) stimulation (1, 2, 3, 4). In
thyrocytes, including functioning rat FRTL-5 cells in continuous
culture, TGF-ß1 inhibits cell proliferation and TSH-induced iodide
uptake, thyroglobulin (TG) biosynthesis, and endothelin production
(5, 6, 7, 8). The role of TGF-ß polypeptides in regulating MHC gene
expression in the thyroid is less clear; however, TGF-ß1-deficient
transgenic mice have increased MHC class I and II levels in many organs
and develop a rapid, wasting, immune disease (9, 10, 11).
Abnormal expression of MHC Class I and II is associated with thyroid
autoimmunity (12, 13). Recent work has shown that aberrant class II,
together with abnormal TSH receptor (TSHR) expression, can induce
autoimmune hyperthyroidism and a Graves-like syndrome in mice,
despite a normal immune system (14). TGF-ß1-induced immune disease
does not develop in class II-deficient animals (15), and TGF-ß1
regulation of class II expression has been linked to conserved proximal
elements of the 5'-flanking region (16). Abnormal class I expression is
also linked to Graves disease, since class I suppression has been
shown to be an important component of the immunosuppressive action of
high (autoregulatory) concentrations of iodide or of methimazole, which
are used to treat Graves disease (13, 17, 18, 19, 20, 21). The mechanism of
TGF-ß1 regulation of MHC class I in thyrocytes is unknown as is the
relationship of such regulation to TGF-ß1-inhibited thyroid growth
and function.
Evidence has accumulated that hormones and growth factors that regulate
FRTL-5 cell growth and function can coordinately decrease MHC class I
expression and may prevent autoimmune thyroid disease (13, 17, 22, 23, 24, 25).
Coordinate regulation of class I expression and thyroid genes important
for cell growth and function has been shown to result from the
interaction of thyroid-restricted and ubiquitous transcription factors
with common cis-elements on the 5'-flanking regions of the
class I and thyroid-restricted genes such as the TSHR (13, 22, 23, 24, 25). It
was of interest, therefore, to document the effect of TGF-ß1 on
thyrocyte MHC class I expression and relate this action to the
regulation of thyroid growth and function in the FRTL-5 thyrocyte by
hormones and other factors.
In this report, we show that TGF-ß1 decreases MHC class I expression
in FRTL-5 thyroid cells and acts transcriptionally within -203 bp of
the start site of the gene. It regulates the interaction of
thyroid-restricted as well as ubiquitous transcription factors with two
cis elements, Enhancer A, -180 to -170 bp, and a
downstream regulatory element (DRE), -127 to -90 bp, whose activity
requires a cAMP response element (CRE)-like sequence, -107 to -100 bp
(13, 23, 24, 25). These same cis elements and/or
trans factors are involved in hormone and iodide control of
genes important for thyroid growth and function, i.e. the
TSHR, TG, thyroid peroxidase (TPO), and the sodium iodide symporter
(NIS).
 |
RESULTS
|
---|
TGF-ß1 Down-Regulates MHC Class I Transcription in Thyrocytes;
Its Action Involves Enhancer A and a Downstream CRE-Containing
Regulatory Element (DRE), -127 to -90 bp
TGF-ß1 significantly reduces MHC class I RNA levels in FRTL-5
rat thyroid cells (Fig. 1A
). The mean
decrease in four separate experiments, when compared with ß-actin RNA
levels (which were unaffected by TGF-ß1), was maximal between 5 and
10 ng/ml, evident within 6 h, optimal by 24, and
nonexistent by 72 h (Table 1
). The class I RNA decrease was
accompanied by a decrease in class I antigen expression in control or
-IFN-treated cells (Fig. 1B
). TGF-ß1 had no effect on cAMP levels
in the cells (data not shown) and its effect was evident in non-TSH
treated cells where cAMP levels are low (Table 1
). We could not
distinguish an immediate early response to TGF-ß1 from a later,
secondary response based on these data.

View larger version (19K):
[in this window]
[in a new window]
|
Figure 1. Effect of TGF-ß1 on Class I mRNA Levels (A)
and on Class I Antigen Expression (B)
FRTL-5 cells were grown to 60% confluency. In panel A, cells
were exposed to 5 ng/ml TGF-ß1 for 12 h, at which time total RNA
was isolated and Northern analysis performed using class I and
ß-actin probes. A representative blot is presented as is the mean
class I/ß actin ratio ± SD from four independent
experiments. Control values are arbitrarily set at 1; the decrease by
TGF-ß1 is significant at P < 0.05. In panel B,
class I antigen levels were measured by fluorescence-activated cell
sorting (FACS) analysis in control cells
(left) or cells treated with 100 U/ml
-interferon for 24 h (right) and in the presence
or absence of 5 ng/ml TGF-ß1 for the last 12 h. The
dashed line represents the Leu-4 background control.
Data are representative of four experiments with similar results.
|
|
View this table:
[in this window]
[in a new window]
|
Table 1. Time and Concentration Effect of TGF-ß1 on
MHC Class I RNA Levels and on Promoter Activity in FRTL-5 Cells Stably
Transfected with the p(-203)Class I-CAT Chimera
|
|
The TGF-ß1-induced decrease in class I RNA levels and antigen
expression reflects a predominantly transcriptional action. Thus,
TGF-ß1 reduced the activity of Class I promoter/chloramphenicol
acetyl transferase (CAT) chimeras in transiently (Fig. 2A
) or stably transfected FRTL-5 cells
(Tables 1
and 2
). The TGF-ß1 effect was
dependent on the TGF-ß1 concentration and the time of treatment, in a
manner similar to the effect of TGF-ß1 on class I RNA levels (Table 1
). The effect on transcription was evident whether cells were
maintained in the presence or absence of TSH (Table 2
).

View larger version (29K):
[in this window]
[in a new window]
|
Figure 2. Effect of TGF-ß1 on the Promoter Activity
of Class I CAT Chimeras Having Different 5'-Extensions
In panel A, FRTL-5 cells were grown to 60% confluency in medium with
TSH (6H medium), shifted to 5H medium containing no TSH for 7 days, and
then shifted again to 6H for 2024 h. Cells were washed, incubated
1 h with 20 µg of the class I-CAT chimera plasmid DNA, 2 µg
pRSV-luciferase, and 250 µg DEAE-dextran. Cells were cultured in 6H
medium for 4048 h, and then maintained therein another 12 h with
or without 5 ng/ml TGF-ß1. CAT activity was measured at that time and
normalized for transfection efficiency. Cell viability was
approximately 89 ± 4% in four separate experiments; data are the
mean of these experiments, performed in triplicate, ± SD.
The class I chimeras were PD1 MHC promoter/CAT constructs having
different 5'-lengths as noted. In panel B, a diagrammatic
representation of the different class I chimeras is presented. Some of
the different regulatory elements in each are noted: a,
the tissue-specific region; b, Enhancer A (Enh A);
c, IRE; d, the 38-bp DRE (24 );
e, the CRE-like site (24 ); f, the CAAT
box. pSV0 is the control vector.
|
|
View this table:
[in this window]
[in a new window]
|
Table 2. Effect of 5 ng/ml TGF-ß1 on MHC Class I
Promoter Activity in FRTL-5 Cells Stably Transfected with Class I-CAT
Chimeras Having Different Lengths of the Class I 5'-Flanking
Region
|
|
Initial experiments localized the site of the TGF-ß1-induced class I
decrease within -127 bp of the start of transcription. Thus, similar
decreases in promoter activity were measured in both transiently (Fig. 2A
) and stably (Table 2
) transfected FRTL-5 cells containing
5'-deletions of an 1100-bp Class I promoter/CAT chimera containing 549
[p(-549)CAT], 203 [p(-203)CAT], and 127 [p(-127)CAT] bp of
5'-flanking sequence from the start of transcription. However, TGF-ß1
activity was markedly decreased in a p(-56)CAT chimera containing 56
bp of 5'-flanking sequence and was not measurable in the promoterless
pSV0 control CAT construct (Fig. 2A
and Table 2
).
Although the 5'-deletion data could not distinguish a difference
in the TGF-ß1-induced decrease in p(-127)CAT and p(-203)CAT
promoter activities because of the error values, involvement of
elements upstream of, as well as within -127 bp, of 5'-flanking
sequence became evident when we mutated individual elements in the
p(-203)CAT chimera: 203MA, 203
CRE, or both together, 203MA
CRE
(Fig. 3A
). Thus, when the activity of the
p(-203) wild-type CAT chimera was compared with the activity of a
-203-bp chimera having the Enhancer A element (-180 to -170 bp)
mutated (p203MA), the Enhancer A mutation caused a significant, but
incomplete, decrease in the TGF-ß1 response (Fig. 3
). This was not
the case if the IFN response element (IRE; -161 to -150 bp) was
deleted (Fig. 3
; p203
IRE), i.e. the effect of mutating
Enhancer A seemed specific.

View larger version (33K):
[in this window]
[in a new window]
|
Figure 3. Effect of TGF-ß1 (A) on Deletion or Substitution
Mutants of Enhancer A, the IRE, or the CRE in the p(-203)class I-CAT
Constructs That Are Diagrammatically Noted in Panel B
Individually isolated clones stably transfected with the wild-type
p(-203) bp class I-CAT chimera or with the p(-203)class I chimera
containing the noted mutation of Enhancer A [p(-203)MA], a deletion
of the IRE [p(-203) IRE], a deletion of the CRE-like sequence
[p(-203) CRE], or both a mutation of Enhancer A plus a deletion of
the CRE [p(-203)MA CRE] were grown to 70% confluency in 6H medium
and then maintained without TSH (5H medium) for 6 days. Cells were then
exposed to 5 ng/ml concentrations of TGF-ß1 for 12 h before CAT
activity was measured. Data are the mean of three experiments,
performed in triplicate, ± SD. A star
denotes a significant P < 0.05 decrease
vs. its untreated control, which has arbitrarily been
set at 100%. Statistical differences between constructs,
P < 0.05, are also noted. The sequences of the
CRE, the IRE, Enhancer A, and mutated Enhancer A are noted on the
bottom.
|
|
Within -127 bp, the TGF-ß1 response was decreased, but not
completely, if the CRE-like sequence (-107 to -100 bp) within the DRE
(26), -127 to -90 bp, was deleted (Fig. 3
; p203
CRE). It appeared,
however, that Enhancer A and the CRE might be functionally
interrelated, since modification of both was required to abolish
TGF-ß1 activity completely (Fig. 3
; p203MA
CRE).
We conclude that TGF-ß1 down-regulates MHC class I transcriptionally
and that its effect involves at least two elements, Enhancer A and the
CRE-like site. The remainder of this report focuses on the effect of
TGF-ß1 on factors interacting with Enhancer A or the CRE-like
sequence, on the relationship of these elements and factors, and on the
basis for their relationships. Since the TGF-ß1 decrease in class I
promoter activity was near maximal 12 h after treatment with 5
ng/ml TGF-ß1, these conditions were used in all subsequent
experiments unless otherwise noted.
The TGF-ß1 Action on Enhancer A Involves Coordinately
Decreased Binding of a fra-2/NF-
B p50 Subunit Heterodimer and
Increased Binding of a p50/p65 NF-
B Subunit Heterodimer; Both
Actions Suppress MHC Class I Gene Expression
Using electrophoretic mobility shift assay (EMSA)
and a 74-bp radiolabeled probe spanning -203 to -130 bp, which
includes both Enhancer A and the IRE (Fig. 4
, bottom), we performed
binding studies under low salt conditions. Cell extracts from
TGF-ß1-treated cells exhibited a significant reduction in a complex
that migrated near the top of the gel (Fig. 4A
, lane 2 vs.
3, arrow) and had characteristics of MOD-1 (23, 25). MOD-1
is a complex between enhancer A and a heterodimer of fra-2 with the p50
subunit of NF-
B (23, 25). MOD-1 is decreased by hydrocortisone (23),
by high concentrations of iodide (25), and by phorbol esters (25), all
of which regulate FRTL-5 cell growth and function (25, 26, 27, 28, 29, 30). A decrease
in MOD-1 has been circumstantially associated with decreased class I
expression, whereas its increase, i.e. by
-IFN, is
associated with increased class I expression (23, 25).

View larger version (46K):
[in this window]
[in a new window]
|
Figure 4. Ability of TGF-ß1 to Decrease Formation of the
MOD-1 Protein/DNA Complex between FRTL-5 Thyroid Cell Extracts and
Enhancer A (A) but Increase Formation of a p50/p65 Heterodimer Complex
with Enhancer A (B)
FRTL-5 thyroid cells were grown to near confluency in 6H medium
containing 5% calf serum and then maintained for 6 days in 5H medium
(no TSH) with 5% calf serum. Cells were fed fresh medium with or
without 5 ng/ml TGF-ß1 and extracts prepared after 12 h. In
panel A, cell extracts were incubated with a radiolabeled 74-bp
fragment of the MHC class I promoter between -203 and -130 bp, termed
the 74 probe and diagrammatically represented in panel C. Incubations
were in a low salt buffer without detergents; EMSAs were used to
identify protein DNA complexes. Lane 1 contains the radioactive probe
alone; lanes 2 and 3 are, respectively, incubations with radioactive
probe and extracts of cells treated with 5 ng/ml TGF-ß1 or from
control cells. In lanes 4 and 5 the incubations with control cells
included a 100-fold excess, over labeled probe, of unlabeled
oligonucleotide with the sequence of mutated Enhancer A or native
Enhancer A, respectively (see Fig. 3 for sequences). The MOD-1 complex
is noted by the arrow and was defined by its ability to be inhibited
and supershifted with antibodies to the p50 subunit of NF- B and to
fra-2 but not control antibodies (23 25 ). In panel B,
nuclear extracts were incubated with the radiolabeled oligonucleotide
containing the Enhancer A sequence of the class I promoter (see Fig. 3 , bottom); incubations were in a high-salt buffer
containing detergent, rather than the low-salt buffer without detergent
used in panel A. Lane 1 is the radioactive probe alone; lanes 2 and 3
are, respectively, the incubations with radioactive probe and extracts
of control cells or cells treated with 5 ng/ml TGF-ß1. Lanes 4 and 5
represent incubations with extracts from cells treated with TGF-ß1
but containing unlabeled oligonucleotide competitors with the wild-type
or mutant Enhancer A, each in a 100-fold excess. Lane 6 notes the
effect of an unlabeled oligonucleotide with the consensus NF- B
binding site, also in a 100-fold excess over probe. Lanes 714 depict
the effect of serum from a normal rabbit or rabbit polyclonal
antibodies to the noted transcription factors. Arrows
denote the location of the TGF-ß1-induced complex identified as a
p50/p65 heterodimer based on the antibody results and the location of a
complex that we suggest is a p50 homodimer based 1) on the antisera
data and 2) on its mobility on a gel relative to complexes formed by
different concentrations of authentic p50 protein (23 25 ).
|
|
To establish that this complex was MOD-1 and interacted with
Enhancer A, we first showed that formation of the complex was decreased
by a 100-fold excess of an oligonucleotide with the sequence of
Enhancer A, but not by the same concentration of the oligonucleotide
with a mutated Enhancer A sequence (Fig. 4A
, lane 5 vs. 4).
The mutated Enhancer A sequence has been separately shown to lose
Enhancer A function and MOD-1 binding (23, 25). Additionally, we showed
that formation of the complex was inhibited and/or supershifted by
specific antibodies against fra-2 or the p50 subunit of NF
B, but not
by antibodies against fra-1, the p65 subunit of NF
B, or other
c-fos family members [data not shown but exactly as
previously described (23, 25)]. We could, therefore, unequivocally
identify this TGF-ß1-decreased complex as MOD-1.
When binding to Enhancer A was simultaneously performed in high-salt
conditions containing detergents, rather than low-salt conditions,
nuclear extracts from TGF-ß1- treated FRTL-5 cells exhibited
increased binding of a p50/p65 heterodimer of NF-
B to Enhancer A
(Fig. 4B
, lane 3 vs. 2), coordinately with decreased MOD-1
binding (Fig. 4A
). Thus, formation of the TGF-ß1-increased complex
was prevented by unlabeled Enhancer A (100x), but not by mutant
Enhancer A at the same concentration (Fig. 4B
, lanes 4 and 5) and was
decreased and/or supershifted by antibodies to the p50 and p65 subunits
of NF-
B (Fig. 4B
, lanes 8 and 9, respectively), but not by
antibodies to c-rel, fra-1, fra-2, c-fos, or
c-jun (Fig. 4B
, lanes 1014). Similar increases in the
p50/p65 heterodimer are induced by iodide and phorbol esters (25).
The complex was decreased by the unlabeled consensus sequence of
NF-
B, 5'-AGTTGAGGGGACTTTCCCAGGC-3' (Fig. 4B
, lanes 6), which contains the core GGGGA sequence of enhancer
A, 5'-TGGGGAGTCCCCGTG-3', but differs in having a
longer inverted repeat (italicized) and a different spacing
to the inverted repeat. Thus, the GGGGA is a critical
element in the formation of the TGF-ß1-induced p50/p65 heterodimer
complex, just as it is for the iodide- or phorbol ester-increased
p50/p65 heterodimer (25).
The decrease in MOD 1 and the increase in p50/p65 heterodimer is
not associated with an ability of TGF-ß1 to affect a change in I
B
in the cytosol or localization of p65 molecules in the nucleus at early
times (Table 3
). Thus, fractionation and
Western blot analysis revealed no significant decrease in I
B or
increase in p65 molecules in the nuclear extract until after 12 h
(Table 3
). Changes in both, however, may contribute at 24 h.
To unequivocally establish the functional relevance of the binding of
MOD-1 or the p50/p65 heterodimer to Enhancer A, we transiently
cotransfected the -203-bp class I-CAT chimera with full length cDNAs
encoding the NF-
B p65 subunit (pMT2T-p65), the NF-
B p50 subunit
(pMT2T-p50), fra-2 (pRSV- fra-2), combinations thereof, or their
control vectors (pMT2T and pRSV). Cotransfection of p50 plus fra-2
markedly increased the promoter activity of the p(-203) class I/CAT
chimera (Fig. 5
, second open
bar). This was not true of their respective control vectors (Fig. 5
, first open bar), of p65 plus fra-2, of either alone, or
of their control vectors (Fig. 5
). In contrast, cotransfection of p50
plus p65 significantly decreased the promoter activity of the p(-203)
class I/CAT chimera (Fig. 5
, fifth open bar). The ability of
TGF-ß1 to decrease class I expression was maintained or relatively
enhanced in the p50/fra-2 cotransfected cells (Fig. 5
, second
black bar) but was lost in the p50/p65 transfected cells (Fig. 5
, fifth black bar). Transfections involving p203MA exhibited
no responses to cotransfections with p50/fra-2 or p50/p65 (data not
shown), unequivocally linking these effects to Enhancer A.
Additionally, the effect of TGF-ß1 on the p50/fra-2 and p50/p65
cotransfections was very specific for the wild-type p(203)
promoter.

View larger version (14K):
[in this window]
[in a new window]
|
Figure 5. Effect of TGF-ß1 on the Transient Expression of
the p(-203) Class I-CAT Chimera That Was Cotransfected with cDNAs
Encoding the p50 or p65 Subunits of NF- B, fra-2, or Control Vectors
Containing These cDNAs, pMT2T and pRSV, Respectively
FRTL-5 cells were grown to 60% confluency in medium with TSH (6H
medium), shifted to 5H medium containing no TSH for 7 days, and then
shifted again to 6H for 2024 h. Cells were washed, incubated 1 h
with 20 µg class I-CAT chimera plasmid DNA, 10 µg of each of the
expression vectors, pMT2T and pRSV, with or without the inserted cDNAs
for p50, p65, and fra-2, 2 µg pRSV-luciferase, and 250
µg DEAE-dextran. Cells were cultured in 6H medium for 4048 h, and
then maintained therein another 12 h with or without 5 ng/ml
TGF-ß1. CAT activity was measured at that time and normalized for
transfection efficiency. Data are the mean of four separate
experiments, performed in triplicate ± SD. Cell
viability was approximately 88 ± 5%. *, Significant
P < 0.05 decrease induced by TGF-ß1 treatment by
comparison to the control cells, which have not been treated with
TGF-ß1. **, A significant decrease in control activity,
P < 0.05, by comparison to cells transfected with
p(-203)CAT plus pMT2T and pRSV control vectors (first open
bar) and whose activity is arbitrarily set at 100%. ***, A
significant increase, P < 0.02, by comparison to
cells transfected with p(-203)CAT plus pMT2T and pRSV control vectors
(first open bar) and whose activity is arbitrarily set
at 100%. ****, A significant P < 0.01 decrease
induced by TGF-ß1 by comparison to an untreated control.
|
|
In sum, the data are the first direct demonstration of the functional
importance of MOD-1 and the p50/p65 heterodimer on class I expression,
of the ability of TGF-ß1 to concurrently decrease their binding to
Enhancer A, and of their importance to the
TGF-ß1-down-regulatory effect on class I. Additional p65 alone, in
cells transfected with pTMT- p65, does not significantly decrease class
I gene expression or enhance TGF-ß1 suppressive activity, suggesting
there is no significant free p50 to form new p50/p65 heterodimer
complexes with enhancer A. The increase in p50/p65 heterodimer may
result from the decrease in MOD-1 complex, releasing p50 already within
the nucleus to interact with p65. The fra-2 would, in this scenario, be
freed to form new complexes; this will be evidenced below. The loss of
the TGF-ß1 suppressive action in p50/p65 transfected cells suggests
that p50/p65 suppression is a dominant effect of TGF-ß1 on Enhancer
A.
TGF-ß1 Decreases the Binding of a Tissue-Specific Transcription
Factor (TTF-1) to the DRE, but Increases the Binding of a Ubiquitous
Transcription Factor (TSEP-1, a Y-box Protein); These Are,
Respectively, an Enhancer and Suppressor of Class I Gene Expression
Whose Activity Requires the CRE-Like Site
TSH and forskolin down-regulate class I gene expression by their
action on the DRE, -127 to -90 bp, whose function depends on the
CRE-like sequence, -107 to -100 bp (24). TGF-ß1-induced
down-regulation of class I also requires the CRE-like site (Fig. 3
).
TSH/forskolin-decreased class I expression is associated with their
ability to induce the formation of new complexes with a class I probe
containing 127 bp of 5'-flanking region (-127 to +1 bp) (24). These
complexes reflect the loss or gain, respectively, of binding to the DRE
by thyroid transcription factor-1 (TTF-1) and the murine homolog of the
human Y-box protein, YB-1, which we had cloned and termed TSHR
suppressor element binding protein-1 (TSEP-1) (24, 31). TTF-1 and
TSEP-1 are tissue-specific and ubiquitous transcription factors,
respectively; they are also, respectively, an enhancer and suppressor
of class I gene expression (24).
TGF-ß1-treatment of FRTL-5 cells maintained without TSH (in 5H
medium) results in the increase of two complexes with the radiolabeled
-127 bp probe (Fig. 6A
, lane 2
vs. 3, Complexes A and B) that have the same migratory
properties as those increased by TSH (Fig. 6A
, lane 4 vs.
3). TGF-ß1-treatment of FRTL-5 cells maintained with TSH (6H medium)
results in a third new complex termed complex C, but no increase in
complexes A and B relative to TGF-ß1-treatment without TSH (Fig. 6A
, lane 5 vs. lane 2). Formation of the C complex is prevented
by including a 150-fold excess of unlabeled oligonucleotide with a
consensus AP-1 site and is inhibited by including anti-c-jun
in the incubation (Fig. 6C
, lanes 2 and 3, respectively, vs.
lane 1).

View larger version (48K):
[in this window]
[in a new window]
|
Figure 6. Ability of TGF-ß1 to Alter the Binding of
Proteins to the Class I DRE, -127 to -90 bp, Whose Activity Is
Dependent on the CRE-Like Site, -107 to -100 bp
FRTL-5 thyroid cells were grown to near-confluency in 6H medium
containing 5% calf serum and then maintained for 6 days in 5H medium
(no TSH) with 5% calf serum. Cells were fed fresh 5H or 6H medium for
24 h and then treated with or without 5 ng/ml TGF-ß1 for 12
h. Whole-cell extracts were prepared as described (Materials and
Methods). In panel A, cell extracts were incubated with a
radiolabeled probe encompassing -127 to +1 bp of the 5'-flanking
region of the class I promoter, termed the -127 probe and
diagrammatically represented at the bottom of the
panel. The DRE, -127 to -90 bp, and its encompassed CRE-like
site, -107 to -100 bp, is noted; its properties were previously
characterized (24 ). Lane 1 contains probe alone, lanes 2 and 3 are
incubations containing probe plus extracts from cells without TSH,
lanes 4 and 5 are plus extracts from cells maintained with TSH. Lanes 3
and 4 are control cell extracts; lanes 2 and 5 are extracts from cells
treated with TGF-ß1. A and B denote complexes increased by TGF-ß1
or TSH treatment. C denotes a complex induced by TGF-ß1 in cells also
treated with TSH. In panel B, EMSAs depict results from incubations
with extracts from cells maintained without TSH but treated with 5
ng/ml TGF-ß1 for 12 h. Incubations in this experiment included a
100-fold excess of unlabeled oligonucleotides representing different
elements: CRE-1, the DRE from -127 to -90 bp (lane 1); the CRE
octamer, -107 to -100 bp, plus 6 bp on either side (lane 2);
oligonucleotide C, the TTF-1/Pax-8 binding site from the TG promoter
(lane 4); and the TSEP-1 binding site from the TSHR promoter (lane 5).
Panel C depicts the effect of including a consensus AP-1
oligonucleotide (lane 2) or an antiserum to c-jun
(Santa Cruz Biotechnology, Inc.) in incubations with the
radiolabeled -127 probe plus extract from cells treated with 5 ng/ml
TGF-ß1 plus TSH. The C complex is noted.
|
|
The similarity of the migration of the A and B complexes formed by the
-127 probe with extracts of TSH or TGF-ß1-treated cells, and the
absence of a significant increase in the intensity of the complexes
formed by extracts from cells treated with both TSH and TGF-ß1,
suggested that the complexes were related and were likely to involve
the DRE, -127 to -90 bp, as previously described (24). That the A and
B complexes in the TGF-ß1-treated cell extracts involved the DRE,
rather than the CRE site alone, was evidenced by the ability of an
oligonucleotide with the sequence of the class I promoter from -120 to
-90 bp (termed CRE-1) to inhibit formation of the TGF-ß1-induced A
and B complexes, but not a homolog that contains only the CRE octamer,
-107 to -100 bp, plus 6 bp on its 3'- or 5'-ends (Fig. 6B
, lanes 1
and 2, respectively, vs. 3). An oligonucleotide containing
CRE-1, with the CRE-like site, -107 to -100 bp, deleted (
CRE),
also did not compete (data not shown). These data indicated that the
TGF-ß1-induced A and B complexes, like the TSH/cAMP-induced A and B
complexes (24), involved binding to the DRE, but also required the
CRE-like site for this binding, consistent with the loss of the
TGF-ß1-activity in p203
CRE, (Fig. 3A
).
EMSA revealed that TSEP-1 was a critical protein in the formation of
the TGF-ß1-induced, as well as the TSH/cAMP-induced A and B complex
(Fig. 6B
). Thus, a single-strand oligonucleotide containing one of the
TSEP-1 binding sites of the TSHR (34),
5'-AAACTACCTCTCAACGCATCCG-3' (-216 to -190 bp in the TSHR
5'-flanking region) inhibited formation of the TGF-ß1-induced A and B
complexes (Fig. 6B
, lane 5 vs. 3). Similar inhibition was
seen using a different single-strand Y-box binding site on the TSHR,
-162 to -140 bp; and no inhibition was evident using a single-strand
oligonucleotide with a mutation in the Y-box protein binding element,
5'-AAACTAGTCTTCAACGCATCCG-3' (italicized and
bold), which had been shown to lose TSEP-1 binding activity (31)
(data not shown). No inhibition was evident using a double-strand
oligonucleotide containing the Pax-8/TTF-1 binding site of the TG
promoter, termed oligo C (32) (Fig. 6B
, lane 4 vs. 3), or a
single-strand oligonucleotide with the sequence of the upstream
SSBP-1/TTF-1 binding site of the TSHR,
5'-CTTGTTGCACGGTGAATTCACGAGAAG-3', -886 to -858 bp in the TSHR
5'-flanking region (33). SSBP-1, Pax-8, and TTF-1 have been shown to
interact with the DRE as well as TSEP-1 (24).
To verify the functional relevance of TSEP-1 for TGF-ß1 action, we
transiently cotransfected the -203-bp class I CAT chimera with
pRc/CMV-TSEP-1 or its control vector pRc/CMV (Fig. 7
). Overexpression of TSEP-1 cDNA
suppressed p(-203)CAT activity (Fig. 7
, last open bar), and
the ability of TGF-ß1 to decrease p(-203)CAT activity was lost (Fig. 7
, last black bar vs. last open bar). Overexpression of the
control vector had no effect on activity with or without TGF-ß1.

View larger version (17K):
[in this window]
[in a new window]
|
Figure 7. Effect of TGF-ß1 on Transient Expression of the
p(-203) class I-CAT Chimera That Was Cotransfected with a cDNAs
Encoding pTSEP-1 cDNA or Its Control Vector, pRc/CMV
FRTL-5 cells were grown to 60% confluency in medium with TSH (6H
medium), shifted to 5H medium containing no TSH for 7 days, and then
shifted back to 6H medium for 24 h. Cells were washed, incubated
1 h with 20 µg of the p(-203) class I-CAT chimera plasmid DNA,
10 µg of each of the pRc/CMV vectors with or without the full-length
TSEP-1 insert (31 ), 2 µg pRSV-luciferase, and 250 µg DEAE-dextran.
Cells were cultured in 6H medium for 12 h, in 5H medium for
40 h, and then with or without 5 ng/ml TGF-ß1 for 12 h. CAT
activity was measured and normalized for transfection efficiency. Data
are the mean of four separate experiments, performed in triplicate
± SD. Cell viability was approximately 89 ±6%. *, A
significant P < 0.05 decrease induced by TGF-ß1
treatment by comparison to the control cells, which have not been
treated with TGF-ß1. **, A loss in the ability of TGF-ß1 to cause a
significant decrease by comparison to control activity as well as a
significant decrease, P < 0.05, in the p(-203)CAT
control by comparison to cells transfected with p(-203)CAT ± the
pRc/CMV control vectors.
|
|
Although the EMSA experiment using the -127-bp probe and competition
with oligo C (Fig. 6B
, lane 4) did not indicate that TTF-1 was involved
in the effect of TGF-ß1 on the DRE, separate results did suggest
TTF-1 involvement (Fig. 8
). Thus,
TGF-ß1 treatment, for the same time and at the same concentration
that was near maximally effective in decreasing class I expression (12
h at 5 ng/ml), significantly (P < 0.05) decreased
TTF-1 RNA and protein levels (Fig. 8A
). TTF-1 protein was measured by
the ability of TGF-ß1 treatment to decrease TTF-1 complex formation
between nuclear extracts and the radiolabeled TSHR TTF-1 site probe
(Fig. 8A
, top), which is TTF-1 specific (33, 34). It was
confirmed by the ability of anti-TTF-1 to completely supershift the
complexes in the control and TGF-ß1-treated extracts (data not
shown).

View larger version (19K):
[in this window]
[in a new window]
|
Figure 8. Effect of TGF-ß1 on TTF-1 RNA levels and TTF-1
Protein Binding to a TTF-1- Specific Binding Site from the TSHR (A), as
Well as Its Effect on TTF-1-Increased p(-203) Class I-CAT Chimera
Activity
In panels A and B, FRTL-5 cells were grown to 60% confluency in medium
with TSH (6H medium), shifted to 5H medium containing no TSH for 7
days, and then treated with 5 ng/ml TGF-ß1 for 12 h. In panel A,
total RNA was isolated and Northern analysis performed using TTF-1 and
ß-actin probes. A representative blot is presented
(top, RNA), as is the mean TTF-1/ß actin ratio ±
SD from four independent experiments
(bottom). Control values for the latter data were
arbitrarily set at 1; *, a significant, P < 0.05,
decrease. In panel A, nuclear extracts were also isolated from the
cells, incubated with a radiolabeled TTF-1-specific probe from the TSHR
(top, protein), and TTF-1 binding measured by EMSA (34 ).
The TSHR TTF-1 element was used in this experiment because it is a pure
TTF-1 binding element which does not interact with Pax-8 (34 ). The
class I DRE has two TTF-1 sites, one upstream and one downstream of the
CRE (Ref. 24 and footnote 1). The upstream TTF-1 site, like oligo C
from the TG promoter, also binds Pax-8 (Ref. 24 and footnote 1). We
could not use the pure downstream class I TTF-1 site because the
functional binding site overlaps the CRE and would give ambiguous
results. The TSHR TTF-1 site is well spaced from the CRE. The
specificity of the complex was established by incubating each with
anti-TTF-1, which eliminated and supershifted the complex (data not
shown). In panel B, FRTL-5 cells were grown to 60%
confluency in medium with TSH (6H medium), shifted to 5H medium
containing no TSH for 7 days, and then shifted back to 6H medium for
24 h. Cells were washed, incubated 1 h with 20 µg of the
p(-203) class I-CAT chimera plasmid DNA, 10 µg of each of the
pRc/CMV vectors with or without the full length TTF-1 (34 ), 2 µg
pRSV-luciferase, and 250 µg DEAE-dextran. Cells were cultured in 6H
medium for 12 h, in 5H medium for 40 h, and then with or
without 5 ng/ml TGF-ß1 for 12 h. CAT activity was measured and
normalized for transfection efficiency. Data are the mean of four
separate experiments, performed in triplicate ± SD.
Cell viability was approximately 91 ± 4%. *, A significant
P < 0.02 decrease induced by TGF-ß1 treatment by
comparison to the control cells, which have not been treated with
TGF-ß1 (first set); **, (last set) denotes a significant decrease of
P < 0.05. In the middle set, ** denotes a
significant increase in control activity, P <
0.02, by comparison to cells transfected with p(-203)CAT alone; and
*** denotes a significant (P < 0.01)
TGF-ß1-induced decrease in class I promoter activity by comparison to
the control cells that were transfected with p(-203)CAT plus
pRc/CMV-TTF-1.
|
|
Consistent with earlier studies (24), when we transiently cotransfected
the -203-bp class I CAT chimera with pRc/CMV-TTF-1 or its control
vector pRc/CMV (Fig. 8B
), overexpression of TTF-1 cDNA increased
p(-203)CAT activity. Overexpression of TTF-1 resulted in the
near-complete loss of TGF-ß1 suppression (Fig. 8B
). Overexpression of
the control vector had no effect on activity with or without TGF-ß1.
In sum, TGF-ß1 also appears to decrease expression and binding of a
tissue-specific enhancer, TTF-1, to the DRE, -127 to -90 bp, as well
as increase the binding of a ubiquitous suppressor, a Y-box protein.
The net result is to suppress the promoter by modulating the activity
of the DRE.
The Two TGF-ß1-Responsive Areas, Enhancer A and the
CRE-Containing DRE, Appear to be Linked by the Action of TGF-ß1 on
c-jun Protein
We explored the possibility that the effects of TGF-ß1 on
Enhancer A and the DRE were interrelated, since modification of both
elements appeared to be required for maximal loss of TGF-ß1 activity
(Fig. 3
). Using the radiolabeled 74-bp probe encompassing
Enhancer A, we performed competition experiments with a 100-fold excess
of unlabeled oligonucleotides encompassing the DRE or reacting with
trans factors binding to the element (24): CRE-1, the
element from -127 to -90 bp (24); the CRE octamer plus 6 bp on either
side (-113 to -94 bp); oligonucleotide C, the TTF-1/Pax-8 binding
site from the TG promoter (32); and the TSEP-1 binding site from the
TSHR promoter between -162 and -140 bp (31) (Fig. 9A
). Conversely, we used a 100-fold
excess of unlabeled oligonucleotides with the sequence of Enhancer A or
the mutated form of Enhancer A to compete for complexes formed with the
radiolabeled -127 probe encompassing the DRE (Fig. 9B
).

View larger version (44K):
[in this window]
[in a new window]
|
Figure 9. MOD-1 Binding in the Presence of Oligonucleotide
Competitors of Protein/DNA Complexes with DRE (A) and the Converse (B),
the Effect of Enhancer A as a Competitor of the Binding of Complexes to
the DRE
FRTL-5 thyroid cells were grown to near confluency in 6H medium and
then maintained for 6 days in 5H medium (no TSH). Cells were fed fresh
5H medium for 24 h and maintained with or without 5 ng/ml TGF-ß1
for 12 h, and whole cell extracts were prepared for EMSA. In panel
A, cell extracts from cells maintained without TSH and untreated with
TGF-ß1 were incubated with a radiolabeled 74-bp probe from -203 to
-130 bp, which includes Enhancer A. Lane 1 contains an incubation with
no unlabeled competitor as a control; however, other incubations
included a 100-fold excess of the following unlabeled oligonucleotides:
the TSEP-1 binding site from the TSHR promoter (lane 2); CRE-1, the
element from -127 to -90 bp, which includes the CRE-like site from
-107 to -100 bp (lane 3); oligonucleotide C, the TTF-1/Pax-8 binding
site from the TG promoter (lane 4); and the CRE octamer, -107 to -100
bp, plus 6 bp on either side (lane 5). The MOD-1 complex is noted by
the arrow and was defined by its ability to be inhibited
and supershifted with antibodies to the p50 subunit of NF- B and to
fra-2 but not control antibodies (data not shown). In panel B, cell
extracts from cells maintained without TSH and untreated with TGF-ß1
(lanes 13) were incubated with a radiolabeled probe encompassing
-127 to +1 bp of the 5'-flanking region of the class I promoter,
termed the -127 probe. Lane 1 contains no competitor; lanes 2 and 3
are incubations containing a 100-fold excess of unlabeled Enhancer A or
unlabeled mutated Enhancer A oligonucleotide (see Fig. 3B , bottom). Lane 4 contains extract from cells treated with
TSH plus TGF-ß1, lane 5 from cells maintained without TSH but treated
with TGF-ß1. Complex C, which is increased by TGF-ß1 treatment of
cells maintained with TSH (lane 4), is noted by an
arrow.
|
|
Oligo TSEP-1 (Fig. 9A
, lane 2) and oligo CRE-1 (Fig. 9A
, lane 3)
inhibited the formation of MOD-1 with the 74-bp probe, but not oligo C
(lane 4) or the CRE octamer plus 6 bp on either side (lane 5). Using
the same extracts with the -127 probe, an excess of an oligonucleotide
with the sequence of Enhancer A, but not mutated Enhancer A, induced
the formation of a complex migrating with the same mobility as the C
complex induced by TGF-ß1 in cells maintained with TSH (Fig. 9B
, lane
2 vs. 4). We concluded, therefore, that the two
TGF-ß1-sensitive regions, the DRE and Enhancer A, were not only
functionally interrelated (Fig. 3
), but also related by interactions
involving common trans factors (Fig. 9
). We wondered whether
this relationship involved c-jun, since complex C appeared
to be related to c-jun (Fig. 6C
). TGF-ß1 can increase
c-jun levels and function in fibroblasts and other cells
(35, 36, 37).
TGF-ß1 treatment of FRTL-5 cells increased c-jun RNA
levels (Fig. 10
). A significant
increase (35 ± 7%) was measurable within 1 h; the maximal
increase was evident by 35 h and continued through 12 h (Fig. 10
). The increase was not evident at 18 h. TGF-ß1-increased
c-jun RNA was associated, therefore, with TGF-ß1-induced
decreases in class I gene expression at early times.

View larger version (68K):
[in this window]
[in a new window]
|
Figure 10. Effect of TGF-ß1 on c-jun RNA
Levels
FRTL-5 cells were grown to 60% confluency in medium with TSH (6H
medium), shifted to 5H medium containing no TSH for 7 days, and then
treated with 5 ng/ml TGF-ß1 for 12 h. Total RNA was isolated and
Northern analysis performed using c-jun and ß-actin
probes (Materials and Methods). A representative blot is
presented; the same results were obtained in four separate experiments.
|
|
We then incubated the -127 radiolabeled probe with a c-jun
homodimer, AP-1, in the absence or presence of extracts from cells
maintained without TSH (Fig. 11
).
Neither recombinant AP-1 nor a control recombinant protein that was
similarly prepared, SSBP-1 (33), was able to bind to the radiolabeled,
double-stranded -127-bp probe in the absence of extract (Fig. 11
, lanes 2 and 3, respectively). However, in the presence of extract from
cells maintained in the absence of TSH and never treated with TGF-ß1,
we could observe the appearance of the C complex in the presence of the
c-jun dimer, AP-1, but not SSBP-1 (Fig. 11
, lanes 5 and 6,
respectively). This suggested c-jun might indeed be relevant
to the TGF-ß1-induced C complex in cells treated with TGF-ß1 as
well as TSH (Fig. 6A
, lane 5).

View larger version (59K):
[in this window]
[in a new window]
|
Figure 11. Recombinant AP-1 (c-jun) Modifies
the EMSA Pattern of Protein/DNA Complexes Formed by the DRE
Recombinant AP-1 or SSBP-1 was incubated with the radiolabeled -127
probe either alone (lanes 23) or with extracts (lanes 56) from
cells maintained without TSH. A new complex is formed with recombinant
AP-1 plus extract, which has the mobility of the C complex interacting
with the DRE (Figs. 6 and 9 ).
|
|
Complexes other than the complex C are increased by the addition of
recombinant AP-1; however, in part, this seems to be an effect of added
protein since the increases in these other complexes are induced by
recombinant SSBP-1 (Fig. 11
, lane 6) or albumin (data not shown). The
increase in the upper complex mimics that observed when unlabeled
enhancer A is added to reactions with the -127 probe plus extract from
cells that are not TSH stimulated (Fig. 9B
, lane 2). We speculate that
increased c-jun, added exogenously (Fig. 11
) or endogenously
by competition with Enhancer A (Fig. 9B
, lane 2; Fig. 13
, model below)
will increase proteins that are components of the upper complex,
i.e. activating transcription factor-1 (ATF-1)
adducts (Ref. 24 ; see below). We have no alternative
explanation for this phenomenon at this time.

View larger version (18K):
[in this window]
[in a new window]
|
Figure 13. Diagramatic Representation of the TGF-ß1 Effect
on Factors Interacting with the Tissue-Specific and Hormone-Sensitive
Regions of the MHC Class I Promoter
The TGF-ß1 effect on c-jun is hypothesized to be a key
component in altering the make up and interactions of the different
complexes existing in the basal state. See text for description.
|
|
In addition, we used a radiolabeled oligonucleotide containing the AP-1
consensus binding site as a probe to measure complexes formed in
extracts from cells maintained without TSH and either treated or not
treated with TGF-ß1 (Fig. 12
).
Extracts from cells that had not been treated with TGF-ß1 had a
single major complex in the presence of the radiolabeled AP-1 consensus
binding site (Fig. 12A
, lane 2, Complex X). Extracts from cells treated
with TGF-ß1 formed a prominent, additional, slower migrating complex
with the radiolabeled AP-1 consensus binding site (Fig. 12A
, lane 3,
Complex Y). Using specific antibodies, we showed that formation of the
slower migrating Y complex was nearly completely inhibited by
anti-c-jun (Fig. 12
, lane 9). It was also nearly completely
inhibited by anti-c-fos (Fig. 12A
, lane 8), significantly
decreased by anti-ATF-1 (Fig. 12A
, lane 7), decreased by
anti-fra-2 (Fig. 12A
, lane 4), but not at all decreased by
antibodies to the p50 and p65 subunits of NF-
B (Fig. 12A
, lanes 5
and 6). In contrast, the faster moving, lower X complex, which was
constitutive in the absence of TGF-ß1 treatment of the cells (Fig. 12A
, lane 2), was predominantly inhibited by anti-ATF-1 (Fig. 12A
, lane
7) and much less dramatically by anti-c-fos or
anti-c-jun (Fig. 12A
, lanes 8 and 9). These data
established that TGF-ß1 treatment increased c-jun RNA and
c-jun binding activity; they established that the increase
in c-jun protein was associated with heterodimers of ATF-1,
c-fos, and fra-2.

View larger version (62K):
[in this window]
[in a new window]
|
Figure 12. The Effect of TGF-ß1 on Protein/DNA Complexes
Formed with a Radiolabeled Oligonucleotide with the Sequence of the
Consensus AP-1 Binding Site
FRTL-5 cells were grown to near confluency in 6H medium and then
maintained 6 days in 5H medium with no TSH. Cells were fed fresh 5H
medium for 24 h and maintained with or without 5 ng/ml TGF-ß1
for 12 h, and whole-cell extracts were prepared for EMSA. In panel
A, cell extracts from cells maintained without TSH and treated (lane 3)
or not (lane 2) with TGF-ß1 were incubated with a radiolabeled
consensus AP-1 probe; probe alone is in lane 1. Lanes 49 contain
extracts from cells maintained without TSH and treated with TGF-ß1,
which were preincubated with the noted antibodies before addition of
the radiolabeled probe. In panel B, the extracts from cells maintained
without TSH but treated with TGF-ß1 were preincubated with a 100-fold
excess of unlabeled oligonucleotides containing the following
sequences: the CRE octamer, -107 to -100 bp, plus 6 bp on either side
(lane 1); CRE-1, the DRE, -127 to -90 bp, which includes the CRE-like
site from -107 to -100 bp (lane 2); and Enhancer A. In Panel C, the
complex formed between AP-1 (c-jun) protein and the
radiolabeled oligonucleotide containing the consensus AP-1 site (lane
1) is compared with the complex in extracts from cells maintained
without TSH and TGF-ß1 (lane 2) and a new complex formed when AP-1
(c-jun) is incubated with the AP-1 consensus binding
site plus the extract from cells maintained without TSH and TGF-ß1
(lane 3). This gel was run for a greater length of time and longer
distance to allow the different complexes to be clearly depicted. The X
and Y complexes were the same in all panels based on parallel
incubations with anti-ATF-1, anti-c-fos, and
anti-c-jun as in panel A.
|
|
That this phenomenon was relevant to complexes formed with Enhancer A
and the DRE was evident in two experiments. First, in competition
experiments, an unlabeled oligonucleotide with the sequence of the DRE
or of Enhancer A could each inhibit formation of both complexes formed
with the AP-1 consensus site (Fig. 12B
, lanes 2 and 3, respectively).
This was not true of the attenuated CRE-site oligo, -113 to -94 bp
(Fig. 12B
, lane 1), which does not prevent formation of
TGF-ß1-induced complexes with the -127 probe. Thus, both Enhancer A
and the DRE interacted with the complexes involving c-jun,
because they competed for the complexes that interacted with the
consensus AP-1 oligonucleotide; their availability for AP-1 complex
formation was enhanced by TGF-ß1. It should be recalled that in the
absence of TGF-ß1 or TSH/cAMP, the MHC class I CRE, as well as the
TSHR CRE, normally form complexes with ATF-1/CREB (cAMP response
element binding protein) (24, 38, 39, 40).
Second, it was noted that addition of AP-1 protein to an extract from
cells maintained without TGF-ß1 (Fig. 12C
, lane 3) resulted in a
complex with the AP-1 consensus binding sequence that migrated not only
more slowly than the AP-1 (c-jun) protein alone (Fig. 12C
, lane 1), but also with a mobility similar to the upper C complex in the
extracts from the TGF-ß1-treated cells, i.e. a phenomenon
that is mimicked by TGF-ß1 treatment, which induces the formation of
complexes involving c-jun, c-fos, ATF-1, and
fra-2.
 |
DISCUSSION
|
---|
Abnormal MHC class I expression has been implicated in autoimmune
thyroid disease (12, 13, 17, 18, 19, 20, 21), whereas TGF-ß1-mediated suppression
of MHC class I has been implicated as a means to mitigate autoimmune
disease (1, 2, 3, 4, 9, 10, 11). The present study shows that TGF-ß1 decreases
MHC class I RNA levels and antigen expression in thyrocytes; more
importantly, it addresses the mechanism by which TGF-ß1
down-regulates MHC class I. We show that the TGF-ß1 suppression of
class I is largely transcriptional and that its effects involve at
least two distinct sequences located within -203 bp of the start of
transcription. We show that the factors and elements involved in
TGF-ß1 regulation of MHC class I are also involved in the hormone
regulation of thyroid-specific genes important for thyroid growth and
function.
The 5'-flanking region of the class I gene has a "hormone-sensitive
region" (-203 to +1 bp) (13, 23, 24, 25, 41) and a "tissue- specific
region" (-771 to -679 bp) (13, 41, 42, 43). Whereas the tissue-specific
region controls constitutive class I expression in different cells
(41, 42, 43), the hormone-sensitive region (HSR) is modulated by cytokines,
growth factors, drugs, and hormones that regulate thyroid cell growth
and function (13, 17, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30). Among these, hydrocortisone (23), iodide
(25), phorbol esters (25), and TSH (10, 17, 24) down-regulate class I
expression, whereas IFN increases expression (13, 17, 23). The HSR is
composed of several regulatory sequences, including Enhancer A (-180
to -170 bp), the IFN-response element or IRE (-161 to -150 bp), and
the DRE, -127 to -90 bp, which contains a CRE-like site, -107 to
-100 bp (13, 17, 23, 24, 25).
We show that TGF-ß1 acts like iodide or phorbol esters to decrease
MOD-1 and increase p50/p65 heterodimer binding to Enhancer A. We
provide transfection data using p50, p65, and fra-2 cDNAs to
unequivocally show that MOD-1 increases, whereas the p50/p65
heterodimer decreases, class I activity. TGF-ß1 thus modulates
transcription factor binding to Enhancer A the same as iodide or
phorbol esters (23, 25), which control thyroid function and growth (23, 25, 26, 27, 28, 29, 30).
The DRE, -127 to -90 bp, whose activity is dependent on a CRE-like
site (-107 to -100 bp), is the main site of action for TSH/cAMP
repression (24). TSH/cAMP decreases formation of a CREB/ATF-1 complex,
which binds to the CRE-like sequence (Ref. 24 and M. Shong, S. I.
Taniguchi, G. Napolitano, M. Saji, M. Ohmori, M. Ohta, H. Shimura, K.
Suzuki, D. S. Singer, and L. D. Kohn, in preparation). They
decrease formation of a complex with a thyroid-restricted transcription
factor, TTF-1, which recognizes sequences flanking each side of the
CRE-like element (Ref. 24 and M. Shong, S. I. Taniguchi, G.
Napolitano, M. Saji, M. Ohmori, M. Ohta, H. Shimura, K. Suzuki, D.
S. Singer, and L. D. Kohn, in preparation). TSH/cAMP also
coordinately increases the ability of a Y-box protein, TSHR suppressor
element protein-1, TSEP-1 (31), to bind the DRE (24). Since TSEP-1 has
a suppressive effect, whereas CREB/ATF-1 and TTF-1 are enhancers, the
net effect of the TSH/cAMP action is a reduction in class I expression
(24). We show that TGF-ß1 regulates the DRE by similarly modulating
the binding of these same factors to the DRE: TSEP-1, TTF-1, and, as a
result of increased c-jun/ATF-1 interactions, very likely
CREB/ATF-1.
One feature of TGF-ß1 action that is not evident in previous studies
with TSH, hydrocortisone, iodide, or phorbol esters appears to be the
interactive involvement of both elements and the induction of a new
complex, complex C, which we uncovered in studies of TGF-ß1-treated
cells that were also maintained with TSH. The mutual interaction of the
two elements is established by cross-competition experiments involving
the ability of oligonucleotides with the sequence of one element to
inhibit the binding properties of the other. We provide evidence that
complex C involves c-jun and show that TGF-ß1 increases
c-jun RNA and protein binding. We provide evidence that
TGF-ß1 action increases c-jun interactions with ATF-1,
c-fos, and fra-2 in EMSA studies using an AP-1 consensus
binding site and that the new complexes interact with both elements,
Enhancer A and the DRE. The effect of TGF-ß1 to increase
c-jun protein levels has been reported in keratinocytes and
fibroblasts (36). c-jun has also been reported to decrease
MHC class I promoter activity at an insulin/serum-modulated element
upstream (35) of the HSR studied herein.
Figure 13
represents a hypothesis of
the dynamic TGF-ß1 regulation of the hormone-responsive region of the
-203 bp 5'-flanking region of class I. Since TGF-ß1 does not
decrease I
B or increase the entrance of NF-
B subunits into the
nucleus at 12 h, when the effect of TGF-ß1 is already near
maximal, we speculate that TGF-ß1-increased c-jun is a
pivotal fulcrum in the relationships between Enhancer A and the DRE
(Fig. 13
). We speculate that TGF-ß1-increased c-jun sets
off an interlocking cascade of reactions that modulates the basal
interactions of Enhancer A, the DRE, and the upstream silencer/enhancer
in the tissue-specific region, -771 to -679 bp, since the latter can
bind c-jun (G. Napolitano, M. Saji, C. Giuliani, S. I.
Taniguchi, M. Shong, V. Montani, K. Suzuki, J. Weissman, D. S.
Singer, and L. D. Kohn, manuscript in preparation). The
increased c-jun RNA and binding activity alter the basal
equilibrium favoring the formation of the MOD-1 (fra-2/p50) complex
with Enhancer A, and instead favor the formation of a new complex
involving c-jun and fra-2, thereby also increasing the
available activated p50 subunit. We speculate that increased
c-jun simultaneously may reduce the interaction of
c-fos and p65 with the upstream silencer/enhancer in the
tissue-specific region controlling class I expression (Fig. 13
and G.
Napolitano, M. Saji, C. Giuliani, S. I. Taniguchi, M. Shong, V.
Montani, K. Suzuki, J. Weissman, D. S. Singer, and L. D.
Kohn, manuscript in preparation. This would contribute c-fos
to the c-jun/fra-2 complex and also increase available p65.
The available p50 and p65 from these actions would allow formation of a
p50/p65 heterodimer able to interact with Enhancer A and suppress class
I expression (Fig. 13
). We suggest that the
c-jun/fra-2/c-fos complex might replace the CREB
interaction with ATF-1 and its interaction with the DRE; CREB/ATF-1
binding to the DRE functions as a class I silencer in FRTL-5 cells
(Refs. 26, 44 and G. Napolitano, M. Saji, C. Giuliani, S. I.
Taniguchi, M. Shong, V. Montani, K. Suzuki, J. Weissman, D. S.
Singer, and L. D. Kohn, manuscript in preparation) (Fig. 13
).
TGF-ß1 additionally increases TSEP-1, but decreases TTF-1 binding to
the DRE (Fig. 13
), thereby increasing the activity of a suppressor
(TSEP-1) and decreasing the activity of an enhancer (TTF-1). The sum of
actions decreases the binding of enhancers of class I expression
(MOD-1, CREB/ATF-1, TTF-1) but increases the binding of suppressors
(p50/p65, Y-box). Both types of modifications contribute to
down-regulation of class I expression.
The Smad family of proteins are mediators of TGF-ß signaling (2, 37, 44, 45, 46, 47, 48). Complexes involving Smad2, Smad3, or Smad 4 can bind directly
to DNA and act as transcription factors (44, 45, 46). Smad binding element
(SBE) consensus sequences have been identified; concatemerization of
these sequences confers Smad 3/4 and TGF-ß responsiveness. Smads can
cooperate with other transcription factors and/or can be mediated by
interactions with FAST-1 (2, 37, 44, 45, 46, 47). Smad transcriptional activity
can also be regulated by binding to coactivators (p300/CBP, AP-1) or
corepressors (Sin3a, Sky, TGIF) (2, 48). The presence of Smad2, Smad3,
and Smad4 and their translocation into the nucleus in response to
TGF-ß has been demonstrated in porcine thyroid cells (49). Smad2 and
Smad4 are expressed in FRTL-5 cells and are functionally active in
enhancing transcription (50).
The ability of TGF-ß1 to alter c-jun levels or binding
activity may be mediated by its effects on Smad proteins and binding
elements (37, 51). Smad proteins can regulate ATF/CREB and NF-
B
family members (37, 53, 54). However, Smad-independent paths exist
(55). Additionally, the basis for the ability of TGF-ß1 to decrease
TTF-1 and increase/or activate TSEP-1 is unclear. Smad/Fast-1 and SBEs
remain to be identified as mediators of TGF-ß effects on these
transcription factors. The ability of TGF-ß1 to decrease TTF-1
levels, as shown by Northern analysis, may separately involve redox
regulation. TGF-ß1 has been reported to have an oxidant effect (56),
which can down-regulate TTF-1 binding to DNA (57). Given the complexity
of the Smad system, it is premature to speculate what role they have in
the regulation described herein. The relationship between Smad proteins
and regulation by CREB, c-jun, ATF family members, or
NF-
B will be complex and remains to be characterized in this
system.
Independent of the role of Smad proteins, which is under investigation,
our observations are consistent with previous studies. TGF-ß1 has
been shown to modulate CREB proteins and ATF-1 (58, 59),
c-jun family proteins (36, 37, 60, 61), and c-fos
family proteins, including fra-2 (61). The coordinated action and
physical interactions of transcription factors from different families
have been widely demonstrated. Indeed, the ATF-1 protein has been
reported to interact with both AP-1 as well as CRE consensus sequences
(60). AP-1 has, in turn, been reported to interact with both
c-fos family members (61) and NF-
B subunits (62). Such
interactions are dependent on structural properties of the proteins
involved; however, homologies between the binding sequences may favor
the interaction. Thus, the CRE-like sequence (TGACGCGA) is similar to
AP-1 consensus binding sequence (TGAc/gTCA) whose core is also present
within the enhancer A region (TGGGGAGTCCCCGTG) and is
similar to the NF-
B consensus sequence
(GGGGACTTTTCCCC).
In sum, we show that TGF-ß1 decreases MHC class I expression by
regulating trans factors interacting with Enhancer A and the
DRE. The TGF-ß1-induced decrease in TTF-1 will decrease TG, TPO, NIS,
and TSHR gene expression (22). The TGF-ß1 effects on MOD-1 and the
p50/p65 heterodimer, like autoregulatory concentrations of iodide (25),
will suppress thyroid growth and function (27, 28, 29, 30). TSEP-1 is a
suppressor of TSHR expression (22, 31); increased TSEP-1 activity
would, therefore, also decrease thyroid growth and function.
 |
MATERIALS AND METHODS
|
---|
Materials
Human Platelet TGF-ß1 was from Sigma (St. Louis,
MO); [
-32P]deoxy-CTP (3000 Ci/mmol),
[14C]chloramphenicol (50 mCi/mmol), and
[
-32P]ATP (3000 Ci/mmol) were from
Amersham Pharmacia Biotech (Arlington Heights, IL).
Antibodies against the p50 and p65 subunits of NF-
B, the
c-fos family, the ATF/CREB family, and c-jun were
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Antiserum and control preimmune serum for rat TTF-1 were obtained from
Dr. S. Kimura (National Cancer Institute, NIH, Bethesda, MD). The AP-1
consensus binding oligo was from Santa Cruz Biotechnology, Inc. The source of all other materials has been detailed (17, 23, 24, 25) or was Sigma.
The TTF-1 expression vector, pRc/CMV-TTF-1, was kindly provided by Dr.
R. Di Lauro (Stazione Zoologica A. Dohrn, Villa Comunale, Naples,
Italy). The pRc/CMV-TSEP-1 was constructed by ligating its full-length
coding sequence with the pRc/CMV vector (Invitrogen, San
Diego, CA) (31). The expression vectors pMT2T-p65 and pMT2T-p50 were
kindly donated by Dr. Ulrich Siebenlist (NIAID, NIH, Bethesda, MD) and
the expression vector, pRSV-fra-2, by Dr. Hideo Iba (University of
Tokyo, Tokyo, Japan).
Cell Culture
FRTL-5 rat thyroid cells (Interthyr Research Foundation,
Baltimore MD; ATCC No. CRL 8305) were a fresh subclone (F1) that had
all properties previously detailed (13, 17, 22, 23, 24, 25, 26, 31, 33, 34, 38, 39, 40, 63, 64). Their doubling time with TSH was 36 ± 6 h; they
were diploid and between their 5th and 25th passage. Cells were grown
in 6H medium consisting of Coons modified F12 (Sigma)
supplemented with 5% calf serum, 1 mM nonessential amino
acids (Life Technologies, Inc., Gaithersburg, MD) and a
mixture of six hormones: bovine TSH (1 x
10-10 M), insulin (10 µg/ml),
cortisol (0.4 ng/ml), transferrin (5 µg/ml),
glycyl-L-histidyl-L-lysine acetate (10 ng/ml),
and somatostatin (10 ng/ml) (63, 64). Fresh medium was added every 2 or
3 days, and cells were passaged every 710 days. In different
experiments, as noted, cells were maintained in 5H medium which
contains no TSH.
RNA Isolation and Northern Analysis
mRNA was isolated using a commercial kit (Quickprep mRNA
Purification Kit, Pharmacia Biotech, Uppsala, Sweden), and
Northern analysis was performed as described (17, 23, 24, 25).
Hybridization was with 1.0 x 106 cpm/ml of
the following cDNA probes: a 1.0-kb HpaI fragment of the MHC
class I (17); ß-actin (kindly provided by Dr. B. Paterson, National
Cancer Institute, NIH, Bethesda, MD); and a 926-bp
HindIII-ApaI TTF-1 cDNA fragment excised from the
pRc/CMV-TTF-1 vector (34). The c-jun probe was from Oncor
(Gaithersburg, MD).
Construction of MHC Class I Promoter-CAT Chimeric Plasmids
Chloramphenicol acetyltransferase (CAT) chimeras of the MHC
class I swine (PD1) 5'-flanking region have been described (23, 24, 25) and
are inserted into the multicloning site of the pSV0-based CAT construct
used as a control in all experiments. They are numbered from the
nucleotide at the 5'-end to +1 bp, the start of transcription. CAT
constructs with mutated MHC class I sequences were created by two-step
recombinant PCR methods (65) as detailed (23, 24, 25). The sequences of all
constructs were confirmed by a standard method (66).
Transfection and CAT Assay
Transient transfection used the Class I promoter/CAT chimeras
and a diethylaminoethyl (DEAE)-dextran procedure (67). Cells were grown
to 60% confluency in 6H medium, shifted to 5H medium for 7 days, and
then shifted again to 6H for 2024 h. Cells were washed twice with
PBS, pH 7.4, and incubated 1 h with 5 ml serum-free medium without
hormones (0H), containing 20 µg class I-CAT chimera plasmid DNA, 2
µg pRSV-luciferase, which was used to measure the efficiency of
transfection (23, 24, 25), and 250 µg DEAE-dextran. Cells were then
exposed to 10% dimethylsulfoxide in PBS for 3 min, washed twice in
PBS, cultured in 6H medium for 4048 h, and maintained therein another
12 h with or without TGF-ß1 as noted. Cell viability was
approximately 80% in all experiments. Overexpression experiments
involving cotransfection with pRc/CMV-TTF-1, pRc/CMV-TSEP-1, pMT2T-p65,
pMT2T-p50, pRSV-fra-2, or their control vectors, pRc/CMV, pMT2T, and
pRSV, included 10 µg of the appropriate plasmid DNA.
In some experiments we used FRTL-5 cells that had been stably
transfected with the pSV0-based CAT-PD1 chimeras as described (23). To
test the effect of TGF-ß1, three individually isolated clones of each
construct were grown to 70% confluency in 6H medium, maintained
without TSH (5H medium) for 6 days, and exposed to TGF-ß1 before CAT
activity was measured. CAT assays were performed as described
(23, 24, 25).
Cell and Nuclear Extracts
FRTL-5 cells were grown in the presence of complete 6H medium
until 60% confluent, and then maintained in 5H medium with 5% calf
serum for 7 days, and finally exposed to TGF-ß1. Cellular extracts
were prepared by a modification of methods described previously (23, 24, 68). In brief, cells were washed twice in cold PBS, pH 7.4,
scraped, and centrifuged (500 x g). The cell pellet
was resuspended in 2 volumes of Dignam buffer C (25% glycerol, 20
mM HEPES-KOH, pH 7.9, 1.5
mM MgCl2, 0.42
M NaCl, 0.5 mM
dithiothreitol, 1 mg/ml leupeptin, 1 µg/ml pepstatin, and 0.5
mM phenylmethylsulfonyl fluoride). The final NaCl
concentration was adjusted on the basis of cell pellet volume to 0.42
M. Cells were lysed by repeated cycles of
freezing and thawing. The extracts were centrifuged (100,000 x
g) at 4 C for 20 min. The supernatant was recovered,
aliquoted, and stored at -70 C.
Nuclear extracts were prepared as described previously (23, 24, 25, 31, 33, 34) from identically treated and harvested FRTL-5 cells. After
centrifugation at 500 x g, the cells were suspended in
5 pellet volumes of 0.3 M sucrose and 2% Tween
40 in Buffer A [10 mM HEPES-KOH, pH 7.9,
containing 10 mM KCl, 1.5
mM MgCl2, 0.1
mM EGTA, 0.5 mM
dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl
fluoride, 2 µg/ml leupeptin, and 2 µg/ml pepstatin A). After
freezing, thawing, and gently homogenizing, nuclei were isolated by
centrifugation at 25,000 x g on a 1.5
M sucrose cushion containing the same buffer.
Nuclei were lysed in Buffer B (10 mM HEPES-KOH,
pH 7.9, 420 mM NaCl, 1.5 mM
MgCl2, 0.1 mM EGTA, 10%
glycerol, 0.5 mM DTT, 0.5
mM phenylmethylsulfonyl fluoride, 2 µg/ml
leupeptin, and 2 µg/ml pepstatin A). After centrifugation at
100,000 x g for 1 h, the supernatant was dialyzed
for use in gel mobility shift analyses using 10
mM Tris-Cl at pH 7.9, 1 mM
MgCl2, 1 mM DTT, 1
mM EDTA, and 5% glycerol, aliquoted, and stored
at -70 C.
Electrophoretic Mobility Shift Assays (EMSAs)
DNA probes were created by restriction enzyme treatment of the
chimeric CAT constructs described above and purified from 2% agarose
gel using QIAEX (QIAGEN, Chatsworth, CA) (23, 24, 25, 31, 33, 34). Oligonucleotides were synthesized by Operon Technologies (Alameda, CA). They were labeled with
[
-32P]dCTP using Klenow or with
[
-32P]ATP using T4 polynucleotide kinase,
and then purified on an 8% native polyacrylamide gel (23, 24, 25, 31, 33, 34).
EMSAs were performed as previously described (23, 25). Binding
reactions in low salts and no detergent included 1.5 fmol
[32P]DNA, 3 µg cell extract, and 1 µg
poly(dI-dC) in 10 mM Tris-Cl, pH 7.9, 1 mM
MgCl2, 1 mM DTT, 1 mM
EDTA, and 5% glycerol in a 20 µl total volume (23, 25). Binding
reactions in high salts plus detergent included 1.5 fmol of
[32P]DNA, 2 µg extract, and 0.5 µg
poly(dI-dC) in 10 mM Tris-Cl, pH 7.9, 5 mM
MgCl2, 50 mM KCl, 1 mM
DTT, 1 mM EDTA, 0.1% Triton X-100, and 12.5% glycerol
(25). Incubations were at room temperature for 30 min. Where indicated,
unlabeled oligonucleotide competitors, recombinant proteins, or
antibodies were added to the binding reaction and incubated with the
extract for 20 min before the addition of labeled DNA. After
incubations, reaction mixtures were electrophoresed on 45% native
polyacrylamide gels at 160 V in 0.5xTBE at room temperature. Gels were
dried and autoradiographed.
Determination of Effects of TGF-ß1 on I
B and NF-
B
Localization and Levels
Measurements of I
B in the cytosol and NF-
B in the nucleus
were adapted from a procedure described previously (69). Cell pellets
were prepared as described above and lysed in boiling 1% SDS
containing 1 mM sodium orthovanadate and 10 mM
Tris, pH 7.4. Nuclear extracts were prepared as above. Fifty micrograms
of cell lysate protein were electrophoresed on 12% SDS-polyacrylamide
gels to measure I
B; 50 µg nuclear extract were electrophoresed on
8% SDS-polyacrylamide gels to measure NF-
B. Proteins were
transferred to polyvinyldifluoride membranes, blocked for 1
h in 5% nonfat dry milk, and then incubated for 1 h with
polyclonal antibodies against p65 or I
B (Rockland Immunochemicals, Gilbertsville, PA). The membrane was washed and
developed using super signal chemiluminescence reagent (Pierce Chemical Co., Rockland, IL)
Other Assays and Statistical Significance
Recombinant proteins were produced using the pET system
(Novagen, Madison, WI) as described previously (31, 33, 34). Protein
concentration was determined using a BCA protein assays kit
(Pierce Chemical Co.); crystalline BSA was the standard.
For fluorescence-activated cell sorter (FACS) analysis, single cell
suspensions were prepared and stained as described (14, 17, 70), using
a class I-specific murine monoclonal antibody. Leu-4 was used as a
background control.
All experiments were repeated at least three times with different
batches of cells. Values are the mean ± SD.
Significance between experimental values was determined by two-way
ANOVA and was P < 0.05 or better when data from all
experiments were considered.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Professor Fabrizio Monaco, Chair of Endocrinology, University "G. DAnnunzio", Palazzina Scuole di Specializzazione, Via dei Vestini, 66100 Chieti, Italy.
Received for publication July 6, 1999.
Revision received January 5, 2000.
Accepted for publication January 21, 2000.
 |
REFERENCES
|
---|
-
Wahl SM 1992 Transforming growth factor beta
(TGF-ß) in inflammation: a cause and a cure. J Clin Immunol 12:6174[Medline]
-
Massague, J 1998 TGF-ß signal transduction. Annu Rev
Biochem 67:753791[CrossRef][Medline]
-
Devajyothi C, Kalvakolanu I, Babcock GT, Vasavada HA, Howe
PH, Ransohoff RM 1993 Inhibition of interferon-
-induced major
histocompatibility complex class II gene transcription by
interferon-ß and type ß1 transforming growth factor in human
astrocytoma cells. J Biol Chem 268:1879418800[Abstract/Free Full Text]
-
Ma D, Niederkorn JY 1995 Transforming growth factor-ß
down-regulates major histocompatibility complex Class I antigen
expression and increases the susceptibility of uveal melanoma cells to
natural killer cell-mediated cytolysis. J Immunol 86:263269
-
Colletta G, Cirafici AM, Di Carlo A 1989 Dual effect of
transforming growth factor ß on rat thyroid cells: inhibition of
thyrotropin-induced proliferation and reduction of thyroid-specific
differentiation markers. Cancer Res 49:34573462[Abstract]
-
Holting T, Zielke A, Siperstein AE, Clark OH, Duh QY 1994 Transforming growth factor-ß1 is a negative regulator for
differentiated thyroid cancer: studies of growth, migration, invasion,
and adhesion of cultured follicular and papillary thyroid cancer cell
lines. J Clin Endocrinol Metab 79:806813[Abstract]
-
Coppa A, Mincione G, Mammarella S, Ranieri A, Colletta G 1995 Epithelial rat thyroid cell clones, escaping from transforming growth
factor ß negative growth control, are still inhibited by this
factor in the ability to trap iodide. Cell Growth Differ 6:281290[Abstract]
-
Tseng YL, Lahiri S, JacksonS, Burman KD, Wartofsky L 1993 Endothelin binding to receptors and endothelin production by human
thyroid follicular cells: effects of transforming growth
factor-ß and thyrotropin. J Clin Endocrinol Metab 76:156161[Abstract]
-
Shull MM, Ormsby IE, Kier AB, Pawlowski S, Diebold RJ, Yin M,
Allen R, Sidman C, Proetzel G, Calvin D, Annunziata N, Doetschman T 1992 Targeted disruption of the mouse transforming growth
factor-ß1 gene results in multifocal inflammatory disease.
Nature 359:693699[CrossRef][Medline]
-
Geiser AG, Letterio JJ, Kulkarni AB, Karlsson S, Roberts AB,
Sporn MB 1993 Transforming growth factor b1 (TGF-ß1) controls
expression of major histocompatibility genes in the postnatal mouse:
aberrant histocompatibility antigen expression in the pathogenesis of
the TGF-ß1 null mouse phenotype. Proc Natl Acad Sci USA 90:99449948[Abstract]
-
Kulkarni A, Huh CG, Becker D, Geiser A, Lyght M, Flanders KC,
Roberts AB, Sporn MB, Ward JM, Karlsson S 1993 Transforming growth
factor ß1 null mutation in mice causes excessive inflammatory
response and early death. Proc Natl Acad Sci USA 90:770774[Abstract]
-
Todd I, Londei M, Pujol-Borrell R, Mirakian R, Feldmann M,
Bottazzo GF 1986 HLA-D/DR expression on epithelial cells: the finger on
the trigger. Ann NY Acad Sci 475:241249[Abstract]
-
Kohn LD, Giuliani C, Montani V, Napolitano G, Ohmori M, Ohta
M, Saji M, Schuppert F, Shong MH, Suzuki K, Taniguchi SI, Yano K,
Singer DS 1995 Antireceptor immunity. In: Raynor D, Champion BR (eds)
Thyroid Autoimmunity, RG Landes Co, Austin, TX, pp 115170
-
Shimojo N, Kohno Y, Yamaguchi K-I, Kikuoka S-I, Hoshioka A,
Niimi H, Hirai A, Tamura Y, Saito Y, Kohn LD, Tahara K 1996 Induction
of Graves-like disease in mice by immunization with fibroblasts
transfected with the thyrotropin receptor and a class II molecule. Proc
Natl Acad Sci USA 93:1107411079[Abstract/Free Full Text]
-
Letterio JJ, Geiser AG, Kulkarni AB, Dang H, Kong L,
Nakabayashi T, Mackall CL, Gress RE, Roberts AB 1996 Autoimmunity
associated with TGF-ß1 deficiency in mice is dependent on MHC
class II antigen presentation. J Clin Invest 98:21092119[Abstract/Free Full Text]
-
Reimold AM, Kara CJ, Rooney JW, Glimcher LH 1993 Transforming
growth factor ß1 repression of the
HLA-DR
gene is mediated by conserved proximal promoter elements.
J Immunol 151:41734182[Abstract/Free Full Text]
-
Saji M, Moriarty J, Ban T, Singer DS, Kohn LD 1992 Major
Histocompatibility Complex Class I gene expression in rat thyroid cells
is regulated by hormones, methimazole and iodide as well as interferon.
J Clin Endocrinol Metab 75:871878[Abstract]
-
Schuppert F, Taniguchi S-I, Schröder S, Dralle H, von
zur Mühlen A, Kohn LD 1996 In vivo and in vitro evidence for
iodide regulation of MHC class I and class II expression in human
thyroid disease. J Clin. Endocrinol Metab 81:36223628
-
Mozes E, Kohn LD, Hakim F, Singer DS 1993 Mice deficient in
expression of MHC class I are resistant to experimental systemic lupus
erythematosus. Science 261:9193[Medline]
-
Singer DS, Kohn LD, Zinger H, Mozes E 1994 Methimazole can
prevent development of disease in an experimental model of systemic
lupus erythematosus. J Immunol 153:873880[Abstract/Free Full Text]
-
Mozes E, Zinger H, Kohn LD, Singer DS 1998 Spontaneous
autoimmune disease in (NZBXNZW) F1 mice is ameliorated by treatment
with methimazole. J Clin Immunol 18:106113[CrossRef][Medline]
-
Kohn LD, Shimura H, Shimura Y, Hidaka A, Giuliani C,
Napolitano G, Ohmori M, Laglia G, Saji M 1995 The thyrotropin receptor.
In: Litwack G (ed) Vitamins and Hormones. Academic Press, San Diego,
CA, vol 50:287384
-
Giuliani C, Saji M, Napolitano G, Palmer LA, Taniguchi SI,
Shong M, Singer DS, Kohn LD 1995 Hormonal modulation of major
histocompatibility complex Class I gene expression involves an enhancer
A-binding complex consisting of Fra-2 and the p50 subunit of NF-kB.
J Biol Chem 270:1145311462[Abstract/Free Full Text]
-
Saji M, Shong M, Napolitano G, Palmer LA, Taniguchi SI, Ohmori
M, Ohta M, Suzuki K, Kirshner SL, Giuliani C, Singer DS, Kohn LD 1997 Regulation of major histocompatibility complex Class I gene expression
in thyroid cells: role of the cAMP response element-like sequence.
J Biol Chem 272:2009620107[Abstract/Free Full Text]
-
Taniguchi SI, Shong M, Giuliani C, Napolitano G, Saji M,
Montani V, Suzuki K, Singer DS, Kohn LD 1998 Iodide suppression of
major histocompatibility class I gene expression in thyroid cells
involves enhancer A and the transcription factor, NF-
B. Mol
Endocrinol 12:1933[Abstract/Free Full Text]
-
Saji M, Kohn LD 1990 Effect of hydrocortisone on the ability
of thyrotropin to increase deoxyribonucleic acid synthesis and iodide
uptake in FRTL-5 rat thyroid cells: opposite regulation of
adenosine 3',5'-monophosphate signal action. Endocrinology 127:18671876[Abstract]
-
Nagataki S, Ingbar SH 1986 Autoregulation: the effect of
iodine. In: Ingbar SH, Braverman LE (eds) The Thyroid. Lippincott,
Philadelphia, pp 319330
-
Becks GP, Eggo MC, Burrow GN 1988 Organic iodine inhibits
deoxyribonucleic acid synthesis and growth in FRTL-5 thyroid cells.
Endocrinology 123:545551[Abstract]
-
Saji M, Isozaki O, Tsushima T, Arai M, Miyakawa M, Ohba Y,
Tsuchiya Y, Sano T, Shizume K 1988 Inhibitory effect of iodide on
growth of rat thyroid (FRTL-5) cells. Acta Endocrinol (Copenh) 119:145151[Medline]
-
Tramontano D, Veneziani BM, Lombardi A, Villone G, Ingbar SH 1989 Iodine inhibits the proliferation of rat thyroid cells in culture.
Endocrinology 136:269282[Abstract]
-
Ohmori M, Shimura H, Shimura Y, Kohn LD 1996 A Y-Box protein
is a suppressor factor which decreases thyrotropin receptor (TSHR) gene
expression. Mol Endocrinol 10:7689[Abstract]
-
Damante G, Di Lauro R 1994 Thyroid-specific gene expression.
Biochim Biophys Acta 1218:255266[Medline]
-
Ohmori M, Ohta M, Shimura H, Shimura Y, Suzuki K, Kohn LD 1996 Cloning of the single strand DNA-binding protein important for maximal
expression and thyrotropin-induced negative regulation of the
thyrotropin receptor. Mol Endocrinol 10:14071424[Abstract]
-
Shimura H, Okajima F, Ikuyama S, Shimura Y, Kimura S, Saji M,
Kohn LD 1994 Thyroid-specific expression and cyclic adenosine
3',5'-monophosphate autoregulation of the thyrotropin receptor gene
involves thyroid transcription factor-1. Mol Endocrinol 8:10491069[Abstract]
-
Howcroft TK, Richards JC, Singer DS 1993 MHC Class I gene
expression is negatively regulated by the proto-oncogene c-jun. EMBO J 12:31633169[Abstract]
-
Mauviel A, Chung KY, Agarwal A, Tamai K, Uitto J 1996 Cell
specific induction of distinct oncogenes of the jun family is
responsible for differential regulation of collagenase gene expression
by transforming growth factor-ß in fibroblast and keratinocytes.
J Biol Chem 271:1091710923[Abstract/Free Full Text]
-
Zhang Y, Feng XH, Derynck R 1998 Smad3 and Smad4 cooperate
with cJun/c-Fos to mediate TGF-ß-induced transcription. Nature
394:909913; Erratum 396:491
-
Ikuyama S, Shimura H, Hoeffler JP, Kohn LD 1992 Role of the
cyclic AMP response element in efficient expression of the rat
thyrotropin receptor. Mol Endocrinol 6:17011715[Abstract]
-
Shimura H, Ikuyama S, Shimura Y, Kohn LD 1993 The cAMP
response element in the rat thyrotropin receptor promoter: regulation
by each decanucleotide of a flanking tandem repeat uses different,
additive, and novel mechanisms. J Biol Chem 268:2412524137[Abstract/Free Full Text]
-
Bifulco M, Perillo B, Saji M, Laezza C, Tedesco I, Kohn LD,
Aloj SM 1995 Regulation of 3-hydroxy-3-methylglutaryl coenzyme A
reductase gene expression: identification and characterization of a
cyclic AMP response element (CRE) in the rat reductase promoter. J
Biol Chem 270:1523115236[Abstract/Free Full Text]
-
Singer DS, Mozes E, Kirshner S, Kohn LD 1997 Role of MHC class
I molecules in autoimmune disease. Crit Rev Immunol 17:463468[Medline]
-
Singer DS, Maguire JE 1990 Regulation of the expression of
Class I MHC genes. Crit Rev Immunol 10:235257[Medline]
-
Weissman JD, Singer DS 1991 A complex regulatory DNA element
associated with a major histocompatibility complex Class I gene
consists of both a silencer and an enhancer. Mol Cell Biol 11:42174227[Medline]
-
Derynk R, Zhang Y, Feng X-H 1998 Smads: transcriptional
activators of TGF-ß responses. Cell 95:737740[Medline]
-
Zawel L, Dai JL, Buckhaults P, Zhou S, Kinzler KW, Vogelstein
B, Kern SE 1998 Human Smad3 and Smad4 are sequence-specific
transcription activators. Mol Cell 1:611617[Medline]
-
Dennler S, Itoh S, Vivien D, ten Dijke P, Huet S, Gauthier JM 1998 Direct binding of Smad3 and Smad4 to critical
TGFß-inducible elements in the promoter of human plasminogen
activator inhibitor-type 1 gene. EMBO J 17:30913100[Abstract/Free Full Text]
-
Zhou S, Zawel L, Lengauer C, Kinzler KW, Vogelstein B 1998 Characterization of human FAST-1, a TGFß and activin
signal transducer. Mol Cell 2:121127[Medline]
-
Janknecht R, Wells NJ, Hunter T 1998 TGF-ß-stimulated
cooperation of Smad proteins with the coactivators CBP/p300. Genes Dev 12:21142119[Abstract/Free Full Text]
-
Franzen A, Piek E, Westermark B, Ten Dijke P, Helden NE 1999 Expression of transforming growth factor ß1, activin A, and
their receptors in thyroid follicle cells: negative regulation of
thyroid growth and function. Endocrinology 140:43004310[Abstract/Free Full Text]
-
Carcamo J, Weiss FM, Ventura F, Wieser R, Wrana JC, Attisano
L, Massague J 1994 Type I receptors specify growth inhibitory and
transcription responses to transforming growth factor ß and activin.
Mol Cell Biol 14:38103821[Abstract]
-
Jonk LJ, Itoh S, Heldin CH, Ten Dijke P, Kruijer W 1998 Identification and functional characterizatiom of a Smad binding
element (SBE) in the JunB promoter that acts as a transforming growth
factor-beta, activin, and bone morphogenetic protein-inducible
enhancer. J Biol Chem 273:2114521152[Abstract/Free Full Text]
-
Sano Y, Tashiro S, Gotoh-Mendeville R, Magkawa T, Iishi S 1999 ATF-2 is a common nuclear target of Smad and TAK1 pathways in
transforming growth factor-ß signaling. J Biol Chem 274:89498957[Abstract/Free Full Text]
-
Topper JN, DiChiara MR, Brown JD, Williams AJ, Falb D, Collins
T, Gimbrone Jr MA 1998 CREB binding protein is a required cofactor for
Smad-dependent, transforming growth factor-ß transcriptional
responses in endothelial cells. Proc Natl Acad Sci USA 95:95069511[Abstract/Free Full Text]
-
Kon A, Vindevoghel L, Kouba DJ, Fujimura Y, Uitto J, Mauviel A 1999 Cooperation between Smad and NF-
B in growth
factor-regulated type VIII collagen expression. Oncogene 18:18371844[CrossRef][Medline]
-
Hocevar BA, Brown TL, Howe PH 1999 TGF-ß induces
fibronectin biosythesis through a c-jun N-terminal kinase dependent,
Smad4-independent pathway. EMBO J 18:13451356[Abstract/Free Full Text]
-
Ohbe M, Shibanuma M, Kuroki T, Nose K 1994 Production of
hydrogen peroxide by transforming growth factor-ß1 and its
involvement in induction of egr-1 in mouse osteoblast cells. J
Cell Biol 126:10791088[Abstract]
-
Kambe F, Nomura Y, Okamato T, Seo H 1996 Redox regulation of
thyroid transcription factors, Pax-8 and TTF-1, is involved in their
increased DNA-binding activities by thyrotropin in rat-thyroid FRTL-5
cells. Mol Endocrinol 10:801812[Abstract]
-
Kramer IM, Koomneef I, de Laat SW, van den Eijnden-van Raaij
AJM 1991 TGF-ß1 induces phosphorylation of the cyclic AMP
responsive element binding protein in ML-CC164 cells. EMBO J 10:10831089[Abstract]
-
Banerjee C, Stein JL, van Wijnen J, Frenkel B, Lian JB, Stein
GS 1996 Transforming growth factor-ß1-responsiveness of the rat
osteocalcin gene is mediated by an activator protein-1 binding site.
Endocrinology 137:19912000[Abstract]
-
Rabbi MF, Saifuddin M, Gu DS, Kagnoff MF, Roebuck KA 1997 U5
region of the human immunodeficiency virus type 1 long terminal repeat
contains TRE-like cAMP responsive elements that bind both AP-1 and
CREB/ATF proteins. Virology 233:235245[CrossRef][Medline]
-
Lee SK, Kim HJ, Na SY, Kim TS, Choi HS, Im SY, Lee JW 1998 Steroid-receptor coactivator 1 coactivates activating
protein-1-mediated transactivations through intreraction with c-jun and
c-fos subunits. J Biol Chem 273:166511654[Abstract/Free Full Text]
-
Denhardt DT 1966 Oncogene-initiated aberrant signaling
engenders the metastatic phenotype: synergistic transcription factor
interactions are targets for cancer therapy. Crit Rev Oncog 7:261291
-
Ambesi-Impiombato FS 1986 Fast-growing thyroid cell strain.
Aug 26, US Patent 4 608 341
-
Kohn LD, Valente WA, Grollman EF, Aloj SM, Vitti P 1986 Clinical determination and/or quantification of thyrotropin and a
variety of thyroid stimulatory or inhibitory factors performed in
vitro with an improved thyroid cell line FRTL-5. Sept 2, US patent
4 609 622
-
Higuchi R 1990 Recombinant PCR. In: Inis MA, Gelfund DH,
Sninsky JJ, White TJ (eds) PCR Protocols: A Guide to Methods and
Applications. Academic Press, San Diego, CA, pp 17783
-
Sanger F, Nicklen S, Coulson AR 1977 DNA sequencing with
chain-terminating inhibitors. Proc Natl Acad Sci USA 74:54635467[Abstract]
-
Lopata MA, Cleveland DW, Sollner-Webb B 1984 High level
expression of a chloramphenicol acetyl tranferase gene by
DEAE-dextran-mediated DNA transfections coupled with a
dimethylsulfoxide or glycerol shock treatment. Nucleic Acids Res 12:57075717[Abstract]
-
Dignam J, Lebovitz R, Roeder R 1983 Accurate transcription
initiation by RNA polymerase II in a soluble extract from isolated
mammalian nuclei. Nucleic Acids Res 11:14751489[Abstract]
-
Aljada A, Ghanim H, Assian E, Mohanty P, Hamouda W Garg R,
Dandona P 1996 Increased I
B expression and diminished nuclear
NF-
B in human mononuclear cells following hydrocortisone injection.
J Clin Endocrinol Metab 84:33863388[Abstract/Free Full Text]
-
Saji M, Moriarty J, Ban T, Kohn LD, Singer DS 1992 Hormonal
regulation of MHC class I genes in rat thyroid FRTL-5 cells: TSH
induces a cAMP-mediated decrease in class I expression. Proc Natl Acad
Sci USA 89:19441948[Abstract]