The Thyrotrope-Restricted Isoform of the Retinoid-X Receptor-
1 Mediates 9-cis-Retinoic Acid Suppression of Thyrotropin-ß Promoter Activity
Bryan R. Haugen,
Nicole S. Brown,
William M. Wood,
David F. Gordon and
E. Chester Ridgway
Division of Endocrinology, Diabetes, and Metabolism Department
of Medicine University of Colorado Health Sciences Center
Denver, Colorado 80262
 |
ABSTRACT
|
---|
TSHß is a subunit of TSH that is uniquely
expressed and regulated in the thyrotrope cells of the anterior
pituitary gland. Thyroid hormone receptors (TR) are known to mediate
T3 suppression of TSHß gene expression at the
level of promoter activity. The role of other nuclear receptors in
regulation of this gene is less clearly defined. Retinoid X receptors
(RXR) are a family of nuclear transcription factors that function both
as 9-cis-retinoic acid (RA) ligand-dependent receptors and
heterodimeric partners with TR and other nuclear receptors. Recently,
the RXR isoform, RXR
, has been identified in the anterior pituitary
gland and found to be restricted to thyrotrope cells within the
pitutiary. In this report, we have further characterized the
distribution of RXR
1, the thyrotrope-restricted isoform of RXR
,
in murine tissues and different cell types. We have found that RXR
1
mRNA and protein are expressed in the TtT-97 thyrotropic tumor, but not
the thyrotrope-variant
TSH cells or somatotrope-derived GH3 cells.
Furthermore, we have studied the effects of RXR
1 on TSHß promoter
activity and hormone regulation in these pituitary-derived cell types.
Both T3 and 9-cis-RA independently suppressed promoter
activity in the TtT-97 thyrotropes. Interestingly, the combination of
ligands suppressed promoter activity more than either alone, indicating
that these hormones may act cooperatively to regulate TSHß gene
expression in thyrotropes. The RXR
1 isoform was necessary for the
9-cis-RA-mediated suppression of TSHß promoter activity
in
TSH and GH3 cells, both of which lack this isoform. RXRß, a
more widely distributed isoform, did not mediate these effects.
Finally, we showed that the murine TSHß promoter region between -200
and -149 mediated a majority of the 9-cis-RA suppression
of promoter activity in thyrotropes. This region is distinct from the
T3-mediated response region near the
transcription start site. These data suggest that retinoids can mediate
TSHß gene regulation in thyrotropes and the thyrotrope-restricted
isoform, RXR
1, is required for this effect.
 |
INTRODUCTION
|
---|
TSH is a glycoprotein hormone that is produced only by the
thyrotrope cells of the anterior pituitary gland (1). TSH contains two
dissimilar, noncovalently associated subunits: the
-subunit, which
is shared among other glycoprotein hormones, and the ß-subunit, which
is functionally unique and has expression limited to thyrotropes. We
have been investigating the cell-specific expression and regulation of
the murine TSHß subunit gene. A number of factors including Pit-1,
Pit-1T, thyroid hormone receptor (TR), and a yet undefined 50-kDa
protein appear essential for TSHß promoter activity and its
regulation in thyrotropes (2, 3, 4, 5, 6). Recently, our group (7) and Sugawara
et al. (8) have studied a retinoid X receptor (RXR) isoform,
RXR
, which is restricted to thyrotropes in the anterior pituitary
gland as well as the thyrotrope-derived tumor, TtT-97. The RXRs belong
to a large family of transcription factors and function as both
9-cis-retinoic acid (RA) ligand-dependent receptors and
heterodimeric partners with thyroid hormone (TR), retinoic acid
receptors, and vitamin D receptors (9). Mangelsdorf et al.
(10) have demonstrated that RXR
and RXRß mRNA are widely expressed
in developing embryo and adult murine tissues, while RXR
mRNA is
more limited in distribution, including abundant expression in the
anterior pituitary. RXR
has been further characterized as two
isoforms, RXR
1 and RXR
2, which are generated by alternate
splicing at the 5'-end of the gene (11). RXR
1 mRNA is restricted to
brain and skeletal muscle. RXR
2 mRNA is found in heart, skeletal
muscle, and liver. Pituitary mRNA has not been examined with an RXR
1
or
2-specific probe.
RXRs are recognized as major TR-associated proteins in many positively
regulated promoter systems (12, 13, 14). The role of RXRs in negative
regulation of promoters such as TSHß, however, is less clear.
Hallenbeck et al. (15) showed that RXRß interfered with
TR
-mediated T3 suppression of a negative thyroid hormone
response element (-24 to -1) of the murine (m)TSHß promoter in
fibroblasts. 9-cis-RA did not have an impact on this effect.
Carr and Wong (16) also showed that RXRß could interfere with both
TR
- and TRß-mediated T3 suppression of a negative
thyroid hormone response element (+11 to +27)of the rat (r)TSHß
promoter in COS cells. This effect was also 9-cis-RA
independent. In contrast, Cohen et al. (17) showed that
RXR
interfered with TRß-mediated suppression of hTSHß promoter
(-20 to +1) activity in JEG-3 cells, but this effect was
9-cis-RA dependent. Studies carried out by these groups used
a small region of the TSHß promoter around the transcription start
site, and none of these groups analyzed effects of the
thyrotrope-restricted RXR
isoform.
Breen et al. (18) recently showed that RA-deficient rats had
increased levels of TSHß mRNA in pituitary extracts. They also showed
that treatment with retinyl palmitate lowered these message levels.
Furthermore, they showed in gene transfer studies that
all-trans-RA suppressed activity of a larger fragment of the
rTSHß promoter (-800 to +150), suggesting that retinoids play a role
in regulation of the TSHß gene.
In this report, we have further characterized the tissue and cell type
distribution of the RXR
isoforms. We have also investigated the role
of RXR
1 in T3- and 9-cis-RA hormone-mediated
regulation of mTSHß promoter activity in thyrotropes and
pituitary-derived cells using a large fragment (-390 to +40) of the
mTSHß promoter that contains elements that mediate both
thyrotrope-specific promoter activity and negative regulation by
T3.
 |
RESULTS
|
---|
RXR Isoform mRNA in Various Cell Types
We first evaluated expression of the RXR isoform mRNAs in the two
thyrotrope-derived cell types, TtT-97 and
TSH, by Northern blot
analysis. Figure 1
shows that while RXR
and RXRß
mRNAs are present in both cell types, RXR
transcripts are abundantly
expressed in TtT-97 thyrotropes, but are virtually undetectable in the
TSH cell line, which no longer expresses the TSHß gene and lacks
T3 regulation (19). Liu and Linney (11) further
characterized two subtypes of RXR
,
1 and
2, which are
generated by alternate splicing at the 5'-end of the gene. We therefore
generated oligonucleotide probes corresponding to the unique
5'-sequences of these two subtypes (Fig. 2
) and repeated
Northern blot analysis on RNA from our thyrotrope-derived cell types,
as well as the pituitary-derived GH3 somatomammotropes and
non-pituitary HeLa cells. Figure 3
shows that only
TtT-97 cells express RXR
1 mRNA, while all three pituitary-derived
cell types contain RXR
2 mRNA. Non-pituitary HeLa cells lack both
RXR
subtypes.
To further analyze for the presence or absence of the RXR
1 mRNA in
various mouse tissues and cell types, we performed sensitive RT-PCR on
total RNA (Fig. 4
). RT-PCR of total RNA from different
murine tissues shows that RXR
1 mRNA is present in pituitary tissue,
and a much smaller amount is present in brain and cardiac tissue. Lung
RNA yeilded no detectable message for RXR
1. TtT-97 RNA gave a
product for RXR
1, while
TSH cell RNA showed a barely detectable
band, suggesting that while RXR
1 message is detectable in
TSH
cells with this sensitive technique, it is at a much lower level than
found in TtT-97 thyrotropes. GH3 mRNA had no detectable RXR
1 mRNA. A
control RT-PCR with glyceraldehyde 3-phosphate dehydrogenase (G3PDH)
showed that relatively similar amounts of RNA were used.

View larger version (29K):
[in this window]
[in a new window]
|
Figure 4. RT-PCR of RNA for RXR 1 in Various Tissues and
Cell Types
Total RNA (2.5 µg) from different murine tissues and cell types
underwent reverse transcription with random hexamers. The sample was
divided into two reactions for PCR using oligonucleotides specific for
RXR 1 or G3PDH over 30 cycles under conditions described in
Materials and Methods. One-tenth of the reaction volume
was size-separated on a 1% agarose gel containing ethidium bromide and
photographed on an UV light source.
|
|
Western Blot Analysis of Nuclear Extracts from TtT-97 and
TSH
Cells
To evaluate for the presence of RXR
1 protein in the
thyrotrope-derived cells, we performed Western blot analysis of nuclear
protein extracts from two separate TtT-97 tumors as well as
TSH
cells. The RXR
1-specific antiserum was kindly provided by Dr. W. W.
Chin. Figure 5
shows that no RXR
1 protein is present
in
TSH cells while two imunoreactive bands are seen in both TtT-97
thyrotrope extracts. The lower band (
50 kDa) corresponds to the form
previously reported by Sugawara et al. (8), while the upper
band may represent a different phosphorylation state, an alternate form
of RXR
1, or a protein that cross-reacts with the RXR
1
antiserum.
Effects of RXR Isoforms on the TSHß Promoter in Pituitary-Derived
Cells
To prove that functional receptors were expressed in transiently
transfected cells, we cotransfected each receptor with a
TREpal-TK-luciferase plasmid (kindly provided by Dr. J. L. Jameson)
into GH3 cells (Fig. 6
). Both RXR
1 and RXRß
stimulated the TREpal element in the presence of the RXR ligand
9-cis-retinoic acid (RA), indicating that functional
receptors are indeed expressed. Interestingly, equal amounts of
transfected plasmids for the RXR isoforms mediated a similar promoter
stimulation, suggesting that functionally equivalent amounts of these
receptors are being expressed.

View larger version (16K):
[in this window]
[in a new window]
|
Figure 6. Stimulation of TREpal TK-Luciferase with
9-cis-RA in the Presence or Absence of RXR Isoforms in
GH3 Cells
Transient transfections were carried out as described in
Materials and Methods. Twenty micrograms of a TREpal
(2x) TK promoter-luciferase promoter DNA with 1 µg of pCMV or
pCMV-RXR isoforms was transfected by electroporation into GH3 cells.
Results are expressed as fold stimulation of promoter activity in the
presence of 2 µM 9-cis-RA
vs. no ligand.
|
|
We then cotransfected RXR
1 or RXRß with the mTSHß (-390 to +40)
promoter-luciferase reporter in GH3 and
TSH cells, both of which
lack the RXR
1 isoform. Neither isoform stimulated the TSHß
promoter in these pituitary-derived cells (data not shown). Addition of
more RXR isoform plasmids (10 µg) also had no stimulatory effect on
the TSHß promoter.
Hormonally Induced Effects of RXR Isoforms on the TSHß Promoter
in Pituitary-Derived Cells
To examine the influence of RXR
1 on T3- and
9-cis-RA-mediated TSHß promoter activity, we first
analyzed the effects of T3, 9-cis-RA, and a
combination of the two on mTSHß promoter activity in TtT-97
thyrotropes that contain RXR
1. As shown in Fig. 7
, T3 (10 nM) suppressed promoter activity by
60%. 9-cis-RA (2 µM) also inhibited promoter
activity by 60%. Interestingly, a combination of T3 and
9-cis-RA suppressed promoter activity more (by 80%), which
was significantly greater (P < 0.05) than either
ligand alone.

View larger version (10K):
[in this window]
[in a new window]
|
Figure 7. Murine TSHß Promoter Activity in TtT-97 Cells in
the Presence of T3 or 9-cis-RA
Transient transfections were carried out as described in
Materials and Methods. Twenty micrograms of mTSHß
(-390 to +40) promoter-luciferase reporter DNA were transfected into
510 million TtT-97 cells. Cells were incubated in the absence or
presence of T3 (10 nM), 9-cis-RA
(2 µM) or both. Results are expressed as percent promoter
activity in the presence of ligand vs. no ligand.
Asterisk denotes a significant difference
(P < 0.05) between the T3/9-cis-RA
group and the other two groups.
|
|
To determine whether RXR
1 is required for 9-cis-RA or
T3 suppression of TSHß promoter activity, we next
examined the effects of T3 and 9-cis-RA on
promoter activity in GH3 somatomammotropes that respond to
T3, but lack RXR
1. These cells do, however, contain
RXR
and RXRß (8). T3 alone suppressed TSHß promoter
activity by approximately 50% (Fig. 8A
). Cotransfection
of RXR
1 caused only a 60% suppression of promoter activity,
indicating that this isoform does not influence the T3
response. Interestingly, cotransfection of RXRß appeared to block
T3-mediated inhibition of TSHß promoter activity, which
was also shown by Hallenbeck and co-workers (15) in mouse fibroblasts
as well as by Carr and Wong (22) in COS cells. Figure 8B
shows that
9-cis-RA alone mediated only a modest suppression of TSHß
promoter activity (20%). Cotransfection of RXR
1 mediated a 55%
suppression of promoter activity with 9-cis-RA, while RXRß
had no effect. These data suggest that RXR
1 is necessary for the
9-cis-RA-mediated suppression of TSHß promoter activity
seen in thyrotropes, and that RXR
1 and RXRß function quite
differently in pituitary-derived cells. The combination of
T3 and 9-cis-RA suppressed TSHß promoter
activity only to a level seen with T3 alone in these GH3
cells (Fig. 8C
). However, cotransfection of RXR
1 augmented this
suppression in the presence of both ligands, while RXRß had no
additional effect. This model suggests that the thyrotrope-restricted
RXR
1 isoform is acting in an isoform-specific manner to mediate
ligand-dependent suppression of TSHß promoter activity in
pituitary-derived cells.
Finally, we examined the effects of T3 and
9-cis-RA on TSHß promoter activity in the
thyrotrope-derived
TSH cells that do not respond to T3
(V. D. Sarapura, unpublished) and lack RXR
1. Like GH3 cells,
TSH
cells contain RXR
and RXRß (Fig. 1
). T3 only modestly
suppressed activity of the TSHß promoter in these cells (Fig. 9A
). Cotransfection of either RXR
1 or RXRß did not
influence this lack of suppression. 9-cis-RA alone modestly
suppressed TSHß promoter activity in these cells (Fig. 9B
), but
cotransfection of RXR
1 mediated a 60% suppression of promoter
activity, which is similar to that observed in GH3 cells.
Cotransfection of RXRß was not different than addition of
9-cis-RA alone. Furthermore, cotransfection of the RXR
2
isoform in these
TSH cells had no effect on promoter activity with
9-cis-RA (data not shown). The combination of T3
and 9-cis-RA behaved in a similar fashion to
9-cis-RA alone (Fig. 9C
). These data suggest that RXR
1
may be the primary RXR isoform required for the 9-cis-RA
suppression of TSHß promoter activity in thyrotropes.
5'-Deletion Mapping of the 9-cis-RA Effect on TSHß
Promoter Activity in Thyrotropes
To identify regions of the TSHß promoter responsible for the
9-cis-RA- mediated suppression of promoter activity in
thyrotropes, we cotransfected 5'-deletion mutants of the TSHß
promoter in the pA3 luciferase plasmid into TtT-97 thyrotropes in the
presence and absence of 2 mM 9-cis-RA. Figure 10A
shows that deletions to -200 had no effect on the
9-cis-RA-mediated suppression (6065%) of promoter
activity. A 5'-deletion of the TSHß promoter between -200 and -149
greatly reduced the 9-cis-RA response while a deletion to
-117 had no further effect, suggesting that the -200 to -149 region
of the mTSHß promoter contains elements responsible for a majority of
the 9-cis-RA-mediated suppression of promoter activity in
thyrotropes. Interestingly, our lab and others have mapped the thyroid
hormone-response region of the TSHß promoter to a different area near
the transcription start site (3, 21, 22, 23). Sequence analysis of the
-200 to -149 promoter region reveals a number of putative hormone
response element half-sites (Fig. 10B
).

View larger version (12K):
[in this window]
[in a new window]
|
Figure 10. 5'-Deletion Mapping of the 9-cis-RA
Effect on TSHß Promoter Activity in Thyrotropes
A, Transient transfections were carried out as described in
Materials and Methods. Twenty micrograms of mTSHß
promoter-luciferase reporter DNA were transfected into 510 million
TtT-97 cells with 1 µg pCMV ßgal. Each 5'-promoter deletion is
listed at the bottom; the 3'-promoter end was kept
constant at +40. Results are expressed as percent promoter activity in
the presence of 2 µM 9-cis-RA
vs. no ligand. B, Murine TSHß promoter sequence
between -202 and -139. Arrows represent potential
hormone response element half-sites.
|
|
 |
DISCUSSION
|
---|
In this report we have analyzed the distribution of RXR
1 in
thyrotrope cells compared with other cell lines and tissues. We have
also characterized a ligand-dependent role for the RXR
1 isoform in
ligand-mediated suppression of the TSHß promoter in pituitary-derived
cells. Our studies show that both thyroid hormone and
9-cis-RA suppress TSHß promoter activity in thyrotropes
that contain both TRs and all three RXR isoforms. Furthermore, these
studies suggest that the suppressive effect of 9-cis-RA on
TSHß promoter activity is dependent on the RXR
1 isoform that is
restricted to thyrotrope cells in the anterior pituitary.
RXRs are recognized as the major TR-associated proteins on many
promoters and belong to a large family of nuclear transcription factors
(24). They function as both 9-cis-RA ligand-dependent
receptors and heterodimeric partners with TRs, retinoic acid receptors,
and vitamin D receptors (9). The essential role for these receptors is
illustrated in studies of targeted disruption of the RXR
(25, 26)
and RXRß (27) genes, resulting in significant developmental
abnormalities and death, particularly in the RXR
-deficient animals.
Mangelsdorf et al. (10) demonstrated that RXR
and RXRß
mRNA were widely expressed in developing embryo and adult murine
tissues, while RXR
mRNA was more restricted in its distribution,
including abundant expression in the anterior pituitary. We
investigated the expression of the RXR isoforms in our
thyrotrope-derived cell types and found that while RXR
mRNA was
highly expressed in TtT-97 thyrotropes, this message was virtually
undetectable by Northern blot analysis and barely detectable by RT-PCR
in
TSH cells, which do not express the TSHß subunit gene and lack
T3 regulation. In a recent report, Sugawara and colleagues
(8) further localized RXR
almost exclusively to the thyrotropes of
the rat anterior pituitary gland. These data suggest that the
thyrotrope-restricted RXR
isoform may play a unique role in
thyrotrope development, mature thyrotrope phenotype, or TSHß gene
regulation by factors such as T3 or retinoic acid.
RXR
exists as two isoforms, RXR
1 and RXR
2 (11), which differ
at the N terminus. RXR
1 mRNA is restricted to brain and skeletal
muscle, while RXR
2 is found in heart, skeletal muscle, and liver. In
this study, we show that RXR
1 is the thyrotrope-restricted isoform.
Testing pituitary-derived cells, we also showed that RXR
1 message is
most prominent in TtT-97 thyrotropes and is barely detectable or
undetectable by the sensitive RT-PCR technique in the
thyrotrope-derived
TSH cells and somatotrope-derived GH3 cells.
Since RXR
1 is restricted to the thyrotropes in the anterior
pituitary gland and is lacking in
TSH and GH3 cells, we used these
pituitary-derived cells to investigate the role of RXR
1 on TSHß
promoter function.
While information regarding TR/RXR interaction on positively regulated
elements is well known (12, 13, 14), data for the role of RXRs in negative
regulation of the TSHß promoter is less clear. Using an
oligonucleotide corresponding to the -24 to -1 region of the mouse
TSHß promoter, Hallenbeck et al. (15) showed that RXRß
interfered with TR
1-mediated T3 suppression in mouse
fibroblasts in a 9-cis-RA ligand-independent manner.
Similarly, Carr and Wong (16) in studies with COS cells using the +11
to +27 and +18 to +27 rTSHß fragments showed that both fragments
could specifically confer T3-mediated suppression of
promoter activity with both TR
and TRß. Addition of RXRß
interfered with this response in a 9-cis-RA
ligand-independent manner. Our data, using a larger TSHß promoter
fragment (-390 to +40), agree with these studies using RXRß. In
contrast, Cohen et al. (17) showed that RXR
interfered
with TRß-mediated T3 suppression of the mTSHß promoter
(-20 to +1) in a 9-cis-RA ligand-dependent manner in JEG-3
cells. They further showed that TRß interacted with the TSHß
promoter (-18 to +37) as a monomer, and that this protein-DNA
interaction was disrupted by addition of RXR
, suggesting that RXR
interferes with TR-mediated T3 suppression of the TSHß
promoter by forming heterodimers in solution, and these protein-protein
interactions are 9-cis-RA dependent. Our results showing
that RXRß interferes with the T3 response in a
ligand-independent manner may differ from these results due to our use
of a larger TSHß promoter fragment or isoform differences between
RXR
and RXRß. TR/RXR synergy on the TRH promoter, which is
negatively regulated by T3, has been recently reported
(28). In both CV-1 and JEG-3 cells, addition of RXR
or RXRß
augmented T3-mediated suppression of the hTRH promoter by
TRß. The authors further demonstrated that TR/RXR heterodimers could
form on a putative thyroid hormone response element half-site,
supporting the concept that TR and RXR can functionally interact on
promoter elements outside the classical DR4.
Because RXR
1 was identified as the thyrotrope-restricted isoform, we
examined the ligand-independent and dependent effects of RXR
1 on a
segment of the mTSHß promoter (-390 to +40) containing the
thyrotrope-specific elements in addition to the region(s) that mediate
response to T3 in pituitary-derived cells. RXR
1 did not
appear to alter promoter activity in a ligand-independent manner in GH3
or
TSH cells, which lack RXR
1 but contain endogenous RXR
2,
suggesting that RXR
1 is not sufficient to stimulate promoter
activity in these cells. While investigating ligand-dependent effects
of RXR isoforms, we found that RXRß interfered with
T3-mediated suppression of TSHß promoter activity in GH3
cells, which is consistent with results by Hallenbeck et al.
(15) and Carr and Wong (16). RXR
, however, did not interfere with
this response in these cells, suggesting functional differences between
the two isoforms. Furthermore, 9-cis-RA had the most
profound suppression of promoter activity in both GH3 and
TSH cells
only in the presence of RXR
, and not with RXRß. These data
strongly suggest that RXR isoform differences play a major role in the
modulation of hormone responses in this system. Finally, we showed that
both T3 and 9-cis-RA can independently mediate
suppression of TSHß promoter activity in TtT-97 thyrotropes. This
combined effect is not as pronounced in GH3 cells, which may be
explained by the overexpression of Pit-1T to stimulate promoter
activity. We have further shown that the promoter region responsible
for the 9-cis-RA effect (-200 to -149) is distinct from
the T3- responsive region identified by others (3, 21, 22, 23).
In summary, our data suggest that both thyroid hormone and retinoids
suppress TSHß promoter activity in thyrotropes. Both ligands appear
necessary for maximal inhibition. The thyrotrope-restricted isoform,
RXR
1, appears to mediate 9-cis-RA suppression of promoter
activity in thyrotropes. Identification of RXR
1 partners and
cis-acting elements responsible for this effect will
increase our understanding of complex interactions of hormones on
regulation of gene expression.
 |
MATERIALS AND METHODS
|
---|
Animals and Cell Culture
All LAF-1 mice used in these studies for the TtT-97 tumors were
treated in accordance with the NIH guidelines on animal use and care.
All protocols were reviewed and approved by the University of Colorado
Health Sciences Center Committee on Use and Care of Animals.
Monolayer cultures of GH3 (ATCC CCL 82.1) and
TSH cells were
maintained in DMEM supplemented with 10% FCS. Replacement with the
same medium containing charcoal-stripped FCS, which was lacking
detectable T4 and T3 levels, was done 48 h
before transfection.
Plasmids
mRXR
, ß, and
cDNAs were generously provided by Dr. R.
M. Evans. A NotI to BstX1 RXRb fragment was gel
purified and subcloned into the NotI site of a pCMV
ß-galactosidase vector (Clontech, Palo Alto, CA) from which the
ß-galactosidase coding region was removed. The RXRß fragment and
vector were blunted with T4 DNA polymerase (Promega, Madison, WI) and
ligated (29). A fragment corresponding to the RXR
1 coding region was
generated by PCR from the plasmid supplied by R. M. Evans using
oligonucleotides corresponding to the start
(5'-GCGGATCCATGTATGGAAATTATTCC-3') and termination sites
(5'-GCGAATTCTCAGGTGATCTGCAGTGGGGT-3') of RXR
1. PCR
conditions were 30 cycles at 94 C for 1 min, 45 C for 1 min, and 72 C
for 1 min. Final PCR extension was at 72 C for 7 min. The gel-purified
fragment was blunted and ligated into the pCMV vector as described for
RXRß. Complementary DNA orientation in the plasmids was verified by
sequencing. TSHß promoter-luciferase reporter constructs and
5'-deletion mutants were prepared as previously described (5).
Northern Blot and RT-PCR Analysis
RNA was prepared from cells and tissues, and polyadenylated RNA
was enriched as previously described (30). Ten micrograms of poly A+
RNA from TtT-97 and
TSH cells were size-separated on a 1% agarose,
6% formaldehyde gel, transferred to a nytran filter, and covalently
bound by UV irradiation (Fischer Biotech, Pittsburgh, PA, 120 mJ). The
filter was hybridized overnight with a
[
32P]dCTP-radiolabeled RXR
HindII (450
bp) fragment, washed, and exposed to radiographic film for 16 h at
-70 C (31). The filter was then washed twice with sterile water at 90
C, rehybridized with a radiolabeled RXR
EcoRV (510 bp)
fragment, washed, and exposed to radiographic film. This procedure was
then repeated with an RXRß NcoI (1400 bp) and a mouse
ß-actin fragment.
Five micrograms of poly A+ RNA from TtT-97,
TSH, GH3, and HeLa cells
(Fig. 2
) were size-separated as above. The subsequent filter was
hybridized with a [
32P]ATP kinase-labeled antisense
oligonucleotide corresponding to the unique RXR
1 5'-sequence
(5'-CCATACATGTTGGCTGCTCAGTT-3'). After washing, the filter was exposed
to radiographic film overnight at -70 C. The probe was removed by
treating the filter with sterile water at 90 C two times and
rehybridized with a labeled antisense oligonucleotide corresponding to
the unique 5'-untranslated RXR
2 sequence
(5'-CAGTGGCCAGTTCCCACAGACCCAGCGC-3'). After washing, the filter was
exposed to film for 3 days at -70 C.
RT-PCR was performed as previously described (4). Briefly, RT-PCR was
carried out by reverse transcription of 2.5 µg of total RNA with
random hexamers (600 ng) and avian myeloblastosis virus reverse
transcriptase (Promega). The product was then divided into three PCR
reactions for RXR
1, and G3PDH. Reactions were carried out with 250
ng of a sense oligonucleotide coresponding to unique sequences for
RXR
1 (5'-GCGGATCCATGTATGGAAATTATTCC-3') and an antisense
oligonucleotide corresponding to the termination sequence of RXR
(5'-GCGAATTCTCAGGTGATCTGCAGTGGGGT-3'). G3PDH PCR was performed using
commercially supplied oligonucleotides (Clontech). PCR reactions were
performed with 2.5U Taq polymerase (Boehringer,
Indianapolis, IN) at 94 C for 1 min, 52.5 C for 1 min, and 72 C for 1
min over 30 cycles. One-tenth of the reaction was size-separated on a
1% agarose gel containing ethidium bromide and exposed to UV light for
photography.
Western Blot Analysis
Western blot analysis was carried out as previously described
(4). Briefly, 20 µg of nuclear protein extracts were size-separated
on a 10% polyacrylamide-SDS gel and transferred to nitrocellulose. The
filter was blocked with 7.5% nonfat milk and hybridized for 1 h
with a rabbit antiserum raised against an oligonucleotide specific for
RXR
1 (kindly provided by W. W. Chin) diluted 1:1000 in PBS (PBS 20
mM
Na2HPO4/NaH2PO4, pH
7.4, 100 mM NaCl) with 0.2% Tween-20. After washing, the
filter was hybridized with a goat anti-rabbit IgG antiserum labeled
with horseradish peroxidase (BRL Life Technologies, Inc., Gaithersburg,
MD) at a 1:4000 dilution in 1% nonfat milk 0.2% Tween-20 PBS. The
filter was then washed, a chemiluminescent assay performed (Amersham
Technologies., Arlington Heights, IL) and exposed to radiographic film
for 2 min.
Transient Transfection Studies
Transient transfection assays have been previously outlined (3, 5). Briefly, 20 µg of the mTSHß (-390 to +40) promoter-luciferase
reporter DNA, 1 µg pCMV-RXR isoform or pCMV empty vector DNA, and 1
µg pCMV ß-galactosidase DNA as an internal control for transfection
efficiency were transfected by electroporation into 510 million
TtT-97, 5 million GH3, or 3 million
TSH cells. Five micrograms of
pCMV-Pit-1T were cotransfected in all GH3 experiments for stimulation
of TSHß promoter activity (5). Cells were incubated at 37 C for
40 h in DMEM with 10% charcoal-stripped FCS and presence or
absence of 10 nM T3 (Sigma Chemical Co., St.
Louis, MO) and/or 2 µM 9-cis-RA (provided by
A. Levin, Hoffman-LaRoche, Nutley, NJ). After harvest, cells were
subjected to freeze-thaw extraction and assayed for both luciferase and
ß-galactosidase activity as previously described (4).
Statistical Analysis
Statistical comparisons of transfection studies were carried out
using Kruskal-Wallis nonparametric testing.
 |
ACKNOWLEDGMENTS
|
---|
We thank the Tissue Culture Core Laboratory for maintainence of
the GH3 and
TSH cells. We would also like to thank Dr. Ronald M.
Evans (Salk Institute, San Diego, CA) for the RXR isoform cDNAs, as
well as Dr. William W. Chin (Brigham and Womens Hospital, Boston, MA)
for the RXR
antiserum. We thank Dr. A. Levin (Hoffman-LaRoche,
Nutley, NJ) for the 9-cis-RA.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Bryan R. Haugen, M.D., B151 4200 East Ninth Avenue, Denver, Colorado 80262.
This work was supported by NIH Grants DK-02331, CA-46934, and DK-36842
as well as a grant from the Thyroid Research Advisory Council (Knoll
Pharmaceutical). This work was also supported by a generous gift from
the Lucille P. Markey Charitable Trust.
Received for publication June 24, 1996.
Revision received December 19, 1996.
Accepted for publication January 7, 1997.
 |
REFERENCES
|
---|
-
Pierce JG, Parsons TF 1981 Glycoprotein hormones:
structure and function. Annu Rev Biochem 50:465495[CrossRef][Medline]
-
Gordon DF, Wood WM, Ridgway EC 1988 Organization and
nucleotide sequence of the gene encoding the beta subunit of murine
thyrotropin. DNA 7:1726[Medline]
-
Wood WM, Kao MY, Gordon DF, Ridgway EC 1989 Thyroid hormone
regulates the mouse thyrotropin beta subunit gene promoter in
transfected primary thyrotropes. J Biol Chem 264:1484014847[Abstract/Free Full Text]
-
Haugen BR, Wood WM, Gordon DF, Ridgway EC 1993 A
thyrotrope-specific variant of Pit-1 transactivates the thyrotropin ß
promoter. J Biol Chem 268:2081820824[Abstract/Free Full Text]
-
Haugen BR, Gordon DF, Nelson AR, Wood WM, Ridgway EC 1994 The
combination of Pit-1 and Pit-1T have a synergistic stimulatory effect
on the thyrotropin ß subunit promoter but not the growth hormone or
prolactin promoters. Mol Endocrinol 8:15741582[Abstract]
-
Haugen BR, McDermott MT, Gordon DF, Rupp CL, Wood WM, Ridgway
EC 1996 Determinants of thyrotrope-specific TSH-beta promoter
activation: cooperation of Pit-1 with another factor. J Biol Chem 271:385389[Abstract/Free Full Text]
-
Haugen BR, Wood WM, Gordon DF, Sarapura VD, Ridgway EC 1994 A thyrotrope-derived cell line which has lost thyroid hormone
regulation lacks TR beta 2 and RXR gamma mRNA. Thyroid 4(S1):S-81
-
Sugawara A, Yen PM, Qi Y, Lechan RM, Chin WW 1995 Isoform-specific retinoid-X receptor (RXR) antibodies detect
differential expression of RXR proteins in the pituitary gland.
Endocrinology 136:17661774[Abstract]
-
Giguere V 1994 Retinoic acid receptors and cellular retinoid
binding proteins: Complex interplay in retinoid signaling. Endocr Rev 15:6179[Medline]
-
Mangelsdorf DJ, Borgmeyer U, Heyman RA, Zhou JY, Ong ES, Oro
AE, Kakizuka A, Evans RM 1992 Characterization of the three RXR genes
that mediate the action of 9-cis retinoic acid. Genes Dev 6:329344[Abstract]
-
Liu Q, Linney E 1993 The mouse retinoid-X receptor-gamma gene:
Genomic organization and evidence for functional forms. Mol Endocrinol 7:651658[Abstract]
-
Murray MB, Towle HC 1989 Identification of nuclear factors
that enhance binding of the thyroid hormone receptor to a thyroid
hormone response element. Mol Endocrinol 3:14341442[Abstract]
-
Lazar MA, Berrodin TJ 1990 Thyroid hormone receptors form
distinct nuclear protein-dependent and independent complexes with a
thyroid hormone response element. Mol Endocrinol 4:16271635[Abstract]
-
Burnside J, Darling DS, Chin WW 1990 A nuclear factor that
enhances binding of thyroid hormone receptors to thyroid hormone
response elements. J Biol Chem 265:25002504[Abstract/Free Full Text]
-
Hallenbeck PL, Phyillaier M, Nikodem V 1993 Divergent effects
of 9-cis-retinoic acid receptor on positive and negative thyroid
hormone receptor-dependent gene expression. J Biol Chem 268:38253828[Abstract/Free Full Text]
-
Carr FE, Wong NC 1994 Characteristics of a negative thyroid
hormone response element. J Biol Chem 269:41754179[Abstract/Free Full Text]
-
Cohen O, Flynn TR, Wondisford FE 1995 Ligand-dependent
antagonism by retinoid x receptors of inhibitory thyroid hormone
response elements. J Biol Chem 270:1389913905[Abstract/Free Full Text]
-
Breen JJ, Matsuura T, Ross AC, Gurr JA 1995 Regulation of
thyroid-stimulating hormone beta-subunit and growth hormone messenger
ribonucleic acid levels in the rat: effect of vitamin A status.
Endocrinology 136:543549[Abstract]
-
Sarapura VD, Wood WM, Gordon DF, Ridgway EC 1992 A cell line
which produces the glycoprotein hormone alpha-subunit contains specific
nuclear factors similar to those present in thyrotropes. Thyroid 2:3138[Medline]
-
Hartong R, Wang N, Kurokawa R, Lazar M, Class CK, Apriletti
JW, Dillman WH 1994 Delineation of three different thyroid
hormone-response elements in promoter of rat sarcoplasmic reticulum Ca
2+ ATPase gene. J Biol Chem 269:1302113029[Abstract/Free Full Text]
-
Wondisford FE, Farr EA, Radovick S, Steinfelder HJ, Moates JM,
McClaskey JH, Weintraub BD 1989 Thyroid hormone inhibition of human
thyrotropin ß-subunit gene expression is mediated by a
cis-acting element located in the first exon. J Biol
Chem 264:1460114604[Abstract/Free Full Text]
-
Carr FE, Kaseem LL, Wong NCW 1992 Thyroid hormone inhibits
thyrotropin gene expression via a position-independent negative
L-triiodothyroinine-responsive element. J Biol Chem 267:1868918694[Abstract/Free Full Text]
-
Bodenner DL, Mroczynski MA, Weintraub BD, Radovick S,
Wondisford FE 1991 A detailed functional and structural analysis of a
major thyroid hormone inhibitory element in the human thyrotropin
beta-subunit gene. J Biol Chem 266:2166621673[Abstract/Free Full Text]
-
Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G,
Umesono K, Blumberg B, Mark M, Chambon P, Evans RM 1995 The nuclear
receptor superfamily: The second decade. Cell 83:835839[Medline]
-
Sucov HM, Dyson E, Gumeringer CL, Price J, Chien KR, Evans RM 1994 RXR alpha mutant mice establish a genetic basis for vitamin A
signaling in heart morphogenesis. Genes Dev 8:10071018[Abstract]
-
Kastner P, Grondona JM, Mark M, Gansmuller A, LeMeur M, Decimo
D, Vonesch JL, Dolle P, Chambon P 1994 Genetic analysis of RXR alpha
developmental function: convergence of RXR and RAR signaling pathways
in heart and eye morphogenesis. Cell 78:9871003[Medline]
-
Kastner P, Mark M, Leid M, Gansmuller A, Chin W, Grondona
JM, Decimo D, Krezel W, Dierich A, Chambon P 1996 Abnormal
spermatogenesis in RXR beta mutant mice. Genes Dev 10:8092[Abstract]
-
Hollenberg AN, Monden T, Flynn TR, Boers M-E, Cohen O,
Wondisford FE 1995 The human thyrotropin-releasing hormone gene is
regulated by thyroid hormone through two distinct classes of negative
thyroid hormone response elements. Mol Endocrinol 9:540550[Abstract]
-
Wang K, Koop BF, Hood L 1994 A simple method using T4 DNA
polymerase to clone polymerase chain reaction products. Biotechniques 17:236238[Medline]
-
Gordon DF, Haugen BR, Sarapura VDS, Nelson AR, Wood WM,
Ridgway EC 1993 Analysis of Pit-1 in regulating mouse TSHß promoter
activity in thyrotropes. Mol Cell Endocrinol 96:7584[CrossRef][Medline]
-
Gordon DF, Wood WM, Ocran KW, Kao MY, Sarapura VD, Ridgway EC 1990 TSH subunit gene promoters from a murine alpha-subunit producing
tumor function normally. Mol Cell Endocrinol 71:93103[CrossRef][Medline]