1 Division of Endocrinology, Department of Medicine, Cedars-Sinai Research Institute- University of California at Los Angeles (UCLA) School of Medicine, Los Angeles 90048; 2 Division of Endocrinology, Charles R. Drew University of Medicine and Sciences- UCLA School of Medicine, Los Angeles 90059; 3 Division of Endocrinology, Department of Medicine, West Los Angeles Veterans Affairs Medical Center, Los Angeles, CA 90073; and 4 Laboratory for Molecular Oncology, Center for Human Genetics, University of Leuven and the Flanders Interuniversity Institute for Biotechnology, B-3000 Leuven, Belgium
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ABSTRACT |
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The prohormone convertases (PCs) PC1 and PC2 are key enzymes capable of
processing a variety of prohormones to their bioactive forms. In this
study, we demonstrated that 6-n-propyl-2-thiouracil (PTU)-induced hypothyroidism stimulated, whereas
triido-L-thyronine (T3)-induced hyperthyroidism
suppressed, PC1 mRNA levels in the rat anterior pituitary. Using 5'
deletions of the human PC1 (hPC1) promoter transiently transfected into
GH3 (a somatotroph cell line) cells, we found that T3
negatively regulated hPC1 promoter activity and that this regulation
required the region from 82 to +19 bp relative to the transcription
start site. Electrophoretic mobility shift assays (EMSAs) using
purified thyroid hormone receptor-
1 (TR
1) and retinoid X
receptor-
(RXR
) proteins and GH3 nuclear extracts demonstrated
that the region from
10 to +19 bp of the hPC1 promoter bound TR
1
as both a monomer and a homodimer and bound TR
1/RXR
as a
heterodimer and multimer. EMSAs with oligonucleotides containing point
mutations of the putative negative thyroid response elements (TREs)
exhibited diminished homodimer and loss of multimer binding. We
conclude that there are multiple novel TRE-like sequences in the hPC1
promoter located from
10 to +19 bp.
posttranslational processing; negative thyroid response element; hypothyroidism; processing enzyme; pituitary; regulation; triiodo-L-thyronine
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INTRODUCTION |
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PROHORMONE CONVERTASES (PCs), members of the mammalian family of the subtilisin-like endoproteases, are thought to be responsible for cleavage of many prohormones at paired basic residues to generate bioactive hormones (43, 55). Among the seven members of the PC family recently cloned, PC1 and PC2 are specifically found in neural and endocrine cells equipped with a regulatory-secretory pathway (43, 55). PC1 and PC2 process a variety of brain and pituitary prohormones, including proopiomelanocortin (POMC), prosomatostatin, provasopressin, proneurotensin, pro-thyrotropin-releasing hormone (TRH), and proenkephalin (33, 43). The important role of PC1 and PC2 in hormonal biosynthesis has been elucidated by studies of mice lacking PC2 and of a patient with defective PC1. Mice with a PC2 knockout have absent proglucagon processing and impaired proinsulin processing, and they were hypoglycemic (10); proenkephalin processing was blocked (20). The patient lacking PC1 had severe childhood-onset obesity, postprandial hypoglycemia, infertility, and low levels of ACTH and cortisol with elevated levels of POMC (16, 38).
In the rat pituitary, PC1 is present primarily in the anterior lobe,
with lower levels in the posterior and intermediate lobes, whereas PC2
is found predominantly in the intermediate lobe (44). Rat
intermediate lobe PC1, PC2, and POMC mRNA levels increased with
treatment with the dopamine antagonist haloperidol and decreased with
the dopamine agonist bromocriptine (2, 3, 6). This regulation is likely to involve the intracellular cAMP pathway (17). Limited studies of the regulation of processing
enzymes by thyroid status have also been performed in the rat
pituitary. Rats made hypothyroid by thyroidectomy or by
6-n-propyl-2-thiouracil (PTU) treatment had an increase in
anterior pituitary PC1, PC2 (6), and peptidylglycine
-amidating monooxygenase (PAM, a processing enzyme involved in
amidation) mRNA levels (39) and a decrease in paired basic
amino acid converting enzyme 4 (PACE4, a more ubiquitously distributed
PC) mRNA levels (21). Rats made hyperthyroid by daily
injection of L-thyroxine (T4) showed decreased anterior pituitary PC1, PC2, and PAM mRNA levels and increased PACE4
mRNA levels (6, 21).
Thyroid hormone (triido-L-thyronine, T3) regulates target gene expression by binding with nuclear thyroid hormone receptors (TRs). T3/TR mediates transcriptional regulation through interactions in the promoter region of target genes bearing consensus DNA sequences, referred to as thyroid hormone response element (TRE) (27). Heterodimerization between TR and retinoid X receptor (RXR) usually augments the ligand-dependent stimulation or repression (27). However, in a few TREs, such as the one in human type 1 deiodinase promoter, RXR-independent mechanisms are involved in thyroid hormone regulation (50).
T3 acts through TRs at the transcriptional level to
regulate many genes such as TRH, thyroid stimulating hormone (TSH),
growth hormone (GH), and -myosin heavy chain (27). We
have previously shown that T3 negatively regulates PC2 in
the rat anterior pituitary and in GH3 cells and localized a region on
the human PC2 promoter that contains putative negative TREs (nTREs)
(30). In this paper, we first studied the in vivo
regulation of PC1 mRNA by thyroid status in rat pituitary. To address
the mechanism of T3 regulation of the PC1 gene, we used GH3
cells. This rat somatotroph cell line expressing endogenous TRs
(14) has been widely used to study T3
regulation of both endogenous (35) and exogenous genes (15). We used hPC1 promoter-luciferase constructs and
electrophoretic mobility shift assays (EMSAs) to identify the region
responsible for the negative regulation by T3. These
studies provide a cellular mechanism for the regulation of pituitary
PC1 levels by thyroid hormone.
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MATERIALS AND METHODS |
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Northern blot analysis of pituitary PC1 mRNA from rats receiving PTU and T3. Adult male Sprague-Dawley rats weighing ~250 g were housed in a light- and temperature-controlled environment and were fed standard laboratory rat chow. Three groups of four animals each were treated as follows. In the control group, rats received drinking water for 2 wk, followed by daily intraperitoneal injection of vehicle for 3 days before being killed. In the hypothyroid group, PTU (0.05%) was added to the drinking water of the rats for 2 wk, and then the rats received daily intraperitoneal vehicle injection for 3 days. In the hyperthyroid group, PTU (0.05%) was added to the drinking water of the rats for 2 wk, and then the rats received daily intraperitoneal injection of T3 (300 µg/kg) for 3 days. Animals were then killed by CO2 administration, and plasma was immediately collected from the neck veins. Plasma TSH was measured by RIA according to the manufacturer's instructions (Diagnostic Products, Torrance, CA). The anterior pituitary was separated from the neurointermediate lobe, and total RNA from individual anterior pituitaries was extracted by means of TRIzol (GIBCO BRL, Gaithersburg, MD). Five micrograms of total RNA were fractionated on a 1.2% denaturing agarose gel and transferred to a GeneScreen Plus hybridization transfer membrane (Du Pont NEN, Boston, MA). The pcDNA3.1(+)/rPC1 construct used for generating the rat PC1 riboprobe was cloned from the BamH I and Bgl II fragment of pCD-BDP (rat PC1) (11) into the BamH I site of pcDNA3.1(+) vector (Invitrogen, Carlsbad, CA) in reverse orientation. The construct was further cut by Nhe I to delete the 3'-end fragment and was self-ligated. Northern blots were carried out as described previously (34). The protocol was conducted according to National Institutes of Health guidelines and was approved by the Institutional Animal Care and Use Committee of Cedars-Sinai Medical Center.
Cell culture. GH3 cells (rat somatotroph cells) and JEG-3 cells (human choriocarcinoma cells) were obtained from the American Type Culture Collection (Rockville, MD) and were maintained at 37°C and 5% CO2 in DMEM with 10% fetal bovine serum (FBS; GIBCO BRL) supplemented with 0.075% sodium bicarbonate, 50 IU/ml penicillin-streptomycin and 0.125 mg/ml Fungizone (amphotericin B; GIBCO BRL). CV-1 (African Green monkey kidney) cells were kindly provided by Dr. Phillip Koeffler (Cedars-Sinai Medical Center) and were grown in MEM supplemented with 10% FBS, 2 mM L-glutamine, 0.11 mg/ml sodium pyruvate, and nonessential amino acids.
Plasmid constructs and luciferase assays.
The human PC1 (hPC1)-luciferase fusion gene expression plasmids were
constructed by subcloning progressively truncated hPC1 promoter with
5'-end at 702,
82, and
18 bp relative to the transcription start
site (TSS) into the polylinker region of the promoterless,
luciferase-encoding pGL2-basic plasmid (Promega, Madison, WI). The
3'-end of all these constructs ended at position +216 bp relative to
the TSS. Smaller hPC1 promoter constructs, containing nucleotides from
82 to +111 and from
82 to +19 bp relative to TSS, were also cloned
into the pGL2-basic plasmid and were used for transfection in GH3
cells. The human TRH (hTRH)- (31) and rat GH
(rGH)-luciferase constructs were generated by subcloning the fragments
from
900 to +54 and
528 to +65 bp relative to TSS, respectively,
into the promoterless pA3-luciferase plasmid. The thymidine
kinase-105 (TK-105)-luciferase construct used as a negative control was
kindly provided by Dr. Lin Pei (Cedars-Sinai Medical Center).
Full-length cDNAs of rat TR
1, TR
2, and TR
1 were cloned into a
pCDM8 expression vector (Invitrogen) and were used for cotransfection
in JEG-3 and CV-1 cells.
Nuclear extract preparation and EMSAs.
Nuclear extracts were prepared from GH3 cells according to Dignam et
al. (7). Protein concentrations in nuclear extracts were
determined by Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). The
region of hPC1 from 82 to +19 bp relative to the TSS was generated by
PCR. Double-stranded oligonucleotides corresponding to the hPC1
promoter sequences
10 to +19 bp (O#1) and
37 to
18 bp
(O#2) relative to the TSS were synthesized by GIBCO BRL and
were radiolabeled using [
-32P]ATP (6,000 Ci/mmol; NEN)
and T4 polynucleotide kinase (GIBCO BRL). EMSAs were
performed as follows. GH3 nuclear proteins (0.5 µg), purified TR
1
(0.01-0.1 µg, Santa Cruz Biotechnology, Santa Cruz, CA), or
RXR
(2.0 µg, BIOMOL Research Laboratories, Plymouth Meeting, PA)
proteins were incubated for 10 min at room temperature in reaction
buffer containing 20 mM HEPES buffer (pH 7.9), 50 mM KCl, 6.25 mM
MgCl2, 0.5 mM EDTA, 10% glycerol, 0.5 mM dithiothreitol, and 2 µg
polydeoxyinosinic-deoxycytidylic acid as a nonspecific competitor.
Subsequently, radiolabeled DNA probes were added, and the incubation
was continued for another 30 min. For competition experiments, binding
mixtures were incubated at room temperature with unlabeled
double-stranded oligonucleotides for 10 min before the addition of the
radiolabeled oligonucleotides. For antibody supershift experiment,
specific antibodies were added to mixtures 10 min after the addition of
the radiolabeled DNA probes. All specific antibodies were obtained from
Santa Cruz Biotechnology. Protein-DNA complexes were analyzed on 5%
nondenaturing polyacrylamide gels at 25°C in 0.5× Tris borate/EDTA
buffer and were visualized by autoradiography.
Statistical analysis. Statistical analyses were performed with the InStat 2.03 program using one-way ANOVA for multiple groups and post hoc Student's t-test (corrected using Dunnett's correction factor) for comparing treatment with control.
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RESULTS |
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Regulation of pituitary PC1 mRNA in
vivo by thyroid status.
T3 regulates many genes in the anterior lobe of pituitary.
To study the regulation of pituitary PC1 by thyroid status,
Sprague-Dawley rats were made hypothyroid by adding PTU to the drinking
water for 2 wk. In another group of rats, thyroid hormone levels were then acutely elevated by intraperitoneal injection of T3 3 days before the rats were killed. The hypothyroidism of PTU-treated rats was confirmed by increased TSH levels, and the hyperthyroidism of
T3-treated rats was confirmed by decreased TSH levels (Fig. 1A). The body weights of
PTU-treated rats were significantly lower than those of the control
animals (data not shown). As shown in Fig. 1B,
administration of PTU resulted in an increase in both the 5.0- and
3.0-kb bands of PC1 mRNA in the anterior pituitary compared with
control animals. T3 supplementation for 3 days after PTU
treatment decreased both bands of PC1 mRNA compared with the rats
receiving PTU followed by vehicle. Quantitation demonstrated that PTU
treatment resulted in a fivefold (P < 0.05) increase in the 3.0-kb band of PC1 mRNA compared with control. Moreover, T3 supplementation after PTU treatment resulted in a
10-fold (P < 0.05) decrease in the 3.0-kb band
compared with that of the rats receiving PTU followed by vehicle (Fig.
1C). Similar results were obtained when the 5.0-kb band of
PC1 mRNA was quantitated (data not shown).
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Regulation of hPC1 promoter by
T3 and 9-cis-RA.
To analyze whether the regulation of PC1 in rat anterior pituitary was
due to the direct effect of thyroid hormone on PC1 gene expression, we
analyzed the hPC1 promoter by transient transfection in GH3 cells. We
used the hPC1 971-bp promoter luciferase construct (33).
The TK-105 construct, the hTRH promoter from
900 to +54 bp
(32), and the rGH promoter from
528 to +65 bp, all
cloned upstream of the luciferase reporter gene, were used as
T3-responsive controls in GH3 cells. As shown in Fig.
2, T3 (10
8 M)
inhibited hPC1 promoter activity by 50% (P < 0.05).
9-cis-RA (10
7 M) alone increased (2.5-fold,
P < 0.05) hPC1 promoter activity, whereas addition of
T3 reversed the stimulation to the basal level [P = nonsignificant (NS) vs. control,
P < 0.05 vs. 9-cis-RA treatment]. Consistent with previous observations (23), T3
and 9-cis-RA treatment resulted in a two- and threefold
stimulation of rGH promoter, respectively. The combination of
9-cis-RA and T3 resulted in an additive effect
in the rGH promoter. In contrast to hPC1 promoter, the hTRH promoter
was not affected by 9-cis-RA alone (P = NS),
and the T3-mediated downregulation of hTRH promoter was not
further affected by the presence of 9-cis-RA. In addition, the TK promoter was not affected by either 9-cis-RA,
T3, or their combinations. Taken together, these results
are similar to the results of T3 and/or 9-cis-RA
regulation on the hPC2 promoter (30) and suggest that
9-cis-RA positive and T3 negative regulation are
unique to these two members (PC1 and PC2) of the prohormone convertase
family.
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Deletion analysis of the hPC1 promoter in
GH3 cells.
To further analyze T3-mediated transcriptional regulation
and characterize the putative TREs within the hPC1 promoter, we performed a series of transient transfection assays in GH3 cells with
progressive 5' deletions of the hPC1 promoter. The results are
expressed as luciferase activity in the presence and absence of
T3 and also as percent repression compared with the control cells (without T3). As shown in Fig.
3, all constructs tested containing the
hPC1 promoter revealed similar T3-mediated suppression, which was 43-58% of the control (without T3) and
demonstrated statistically significant suppression. Further
localization of the region on the hPC1 promoter responsible for the
negative regulation by T3 was achieved by deleting the 3'
end of the construct to +19 bp. Thus the minimal construct tested (82
to +19 bp) still exhibited both basal luciferase activity and
downregulation by T3. This suggests that the nTREs are
likely located near the TSS, and the sequence between
82 and +19 bp
is sufficient to mediate the T3-inhibitory effect on the
hPC1 promoter. Similar inhibition by T3 was obtained using
the hTRH promoter (32). In contrast, T3 caused
a twofold stimulation of rGH promoter, as previously reported
(4). No significant influence of T3 on the
control TK- or pGL2-basic luciferase promoter activity was observed,
and the activity of pGL2 basic plasmid was minimal.
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TR isoforms on the
T3-repressive effect of hPC1
promoter in JEG-3 and CV-1 cells.
To study whether T3-/TR-mediated repression of hPC1
promoter activity in GH3 cells is TR isoform specific, we performed
cotransfection experiments with the use of JEG-3 and CV-1 cells, both
of which are known to express minimal amounts of endogenous TRs
(40). Successful transfection of the TR isoforms was
assessed in both cell lines by showing that T3 stimulated
the GH gene and suppressed the TRH gene (data not shown). As shown in
Fig. 4, cotransfected TR was required for
T3-mediated repression of the hPC1 promoter in the two cell
lines tested. However, unlike the results from hTRH (32)
and mouse TRH (40) promoters, cotransfection of TR alone
did not stimulate basal hPC1 promoter activity. Addition of 10 nM
T3 in the cells cotransfected with either TR1 or TR
1 resulted in a 50-70% reduction in luciferase activity, with
similar inhibition in CV-1 and JEG-3 cells. In contrast, addition of 10 nM T3 to the cells cotransfected with TR
2 [the TR
isoform lacking ligand binding capacity (27)] alone did
not affect the hPC1 promoter activity. These results suggest that
T3-mediated hPC1 gene expression is dependent on the
presence of functional TRs and that there is no TR
1 or TR
1
isoform preference in the regulation of hPC1 promoter in the transient
transfection system used in our study.
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Binding of purified TR1 and RXR
proteins to the hPC1
82- to +19-bp
fragment in EMSAs.
To examine whether TR binding sites reside within the functionally
defined fragment from
82 to +19 bp of hPC1 promoter, EMSAs were
carried out using purified recombinant TR
1 and RXR
proteins. Binding was assessed with the hPC1 promoter (from
82 to +19 bp) and
the direct-repeat 4 (DR4) element, which was used as a positive control
(12). As shown in Fig.
5, purified TR protein bound to DR4
(A) and hPC1 (B) as two bands corresponding to TR
monomer (M) and homodimer (HOD). The binding was specific as it was
competed by addition of 100-fold molar excess of unlabeled
identical oligonucleotides [Fig. 5, DR4, lane 3 (A), PC1, lane 3 (B)], but not
by 200-fold molar excess of nonspecific competitor (NON, lane
4). The binding to DR4 was decreased and the binding to hPC1 was
not seen when the concentration of TR
1 was decreased from 0.1 µg
(lane 2) to 0.01 µg (lane 5). RXR alone
(lane 9) at a concentration approximately fivefold more than
the highest concentration of TR
1 (lane 2) did not show
any binding to either DR4 or hPC1 fragments. RXR was then added to a
low concentration of TR
1 (0.01 µg) to show the interaction between
TR and RXR on both DR4 and hPC1 fragments (lanes 6-8).
A strong TR/RXR heterodimer band (HED) was detected for both DR4 and
hPC1 (lane 6). This band has slightly faster mobility than
TR homodimer due to the smaller molecular mass of purified RXR (51 kDa)
than TR (73 kDa) protein. Interestingly, a large-sized multimer band of
TR/RXR was observed by use of the hPC1 fragment (Fig. 5B,
lane 6). The multimer- (for hPC1) and heterodimer-containing
(for both DR4 and hPC1) complexes were supershifted by the addition of
anti-TR and anti-RXR antibody (lanes 7 and 8,
respectively), confirming the identity of the binding complexes.
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EMSAs with two oligonucleotides selected from 82
to +19 bp of the hPC1 promoter.
To further characterize the TR binding region of the hPC1 promoter, two
oligonucleotides containing TRE-like sequences were selected (Fig.
6A) from
10 to +19 bp
(O#1) and from
37 to
18 bp (O#2) from the
82- to +19-bp region of the hPC1 promoter. O#1 contained
one putative TRE half-site that matches six of six nucleotides of the
hexameric consensus TRE half-sites [(A/G)GGT(C/G)A], one
putative TRE half-site matching five of six nucleotides of the
hexameric consensus, and two putative TRE half-sites matching four of
six nucleotides of the hexameric consensus; O#2 contained one putative TRE half-site matching five of six nucleotides of the
consensus TRE. EMSAs were performed with these probes by use of
increasing amounts of purified TR
1. As indicated in Fig.
6B, only minimal binding was detected using O#2,
which contains a single TRE-like half-site (AGGTAA) arranged in reverse
orientation (from 3' to 5'). The lack of binding of this
oligonucleotide (
37 to
18 bp) is not surprising considering that
the
18- to +216-bp construct did not show a decline in inhibition by
T3 compared with the
82- to +216-bp construct. In
contrast, both monomer and homodimer binding were observed by means of
O#1 with increasing intensity of binding when increasing
amounts of TR
1 protein were included (Fig. 6B).
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EMSAs using wild-type and mutant oligonucleotides
with TR1 and RXR
proteins.
To further investigate whether TR/RXR interaction occurred in
O#1, EMSAs were carried out using purified TR
1 and RXR
proteins with wild-type (WT) and mutant (Mut) oligonucleotides as
probes (Fig. 7A). As indicated
in Fig. 7B, when higher amounts of TR
1 were added to the
reactions, both TR monomer and homodimer were detected (lane
2) with O#1. The addition of T3 diminished
the homodimer binding (lane 3) more than the monomer
binding, which is consistent with the observation on other consensus
TRE sequences (9). The addition of TR
1 antibody
supershifted the binding (lane 4), which suggests the
specificity of the TR
1 and O#1 interaction. In the lane
(lane 5) with a low (0.01 µg) amount of TR
1, faint TR
homodimer with a larger amount of monomer binding was detected. RXR
itself did not bind to O#1 (lane 6).
Interestingly, intense TR/RXR heterodimer and multimer binding was
detected (lane 7) using O#1. The addition of TR
antibody (lane 9) and to a lesser extent, T3
(lane 8), reduced the intensity of the heterodimer- and
multimer-containing complexes. The T3 suppression of the
hPC1 heterodimerization is unusual compared with previous reports
(53). These results indicated that not only the TR monomer
and homodimer, but also TR/RXR heterodimer and multimer, can be formed
with the O#1. In contrast to WT O#1, Mut
O#1 (Fig. 7C) lost homodimer binding and
exhibited decreased monomer binding (Fig. 7C, lanes 2 and 5). When TR
1 and RXR were added to the
reactions, heterodimer binding, but not multimer binding, was observed
(Fig. 7C, lane 7).
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Mutation analysis of putative nTRE on the
hPC1 promoter.
To characterize the functional role of these novel, putative nTREs on
the hPC1 promoter, transient transfection analysis in GH3 cells with
the WT and Mut construct (changing the central two and three
nucleotides of two putative nTREs) (see Fig. 7A) constructs
from the 82- to +19-bp hPC1 promoter was performed, and the effect of
T3 was measured. As shown in Fig. 7D,
T3 treatment resulted in a 50% reduction of WT PC1
promoter-luciferase activity, as anticipated. When the Mut construct
was tested, the T3 downregulation of hPC1 promoter activity
was only partially (33%) abolished . These results suggest that the
T3 inhibitory effect may involve more than two TRE-like
half-sites, which is supported by the presence of multimer binding
using this region in the EMSA assay (Fig. 7B, lane
7).
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DISCUSSION |
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The effects of thyroid hormone are protean. In the pituitary, thyroid hormone directly or indirectly regulates gene expression in all cell types (45, 46). The prohormone convertases, including PC1, are important processing enzymes that determine the ratios of many inactive prohormone precursors to active hormones (33, 43). In this study, we demonstrate that changes in thyroid status alter anterior pituitary PC1 mRNA levels and that this regulation is due to the interaction between T3 and TR with nTREs in the hPC1 promoter.
Stimulation of PC1 mRNA by intracellular protein kinase C and protein
kinase A pathways has been demonstrated (17, 18, 51). The
promoter region of hPC1 contains one cAMP-response element (CRE)
consensus sequence (TGACGTCA) and one CRE-like sequence (TGACGTGT) at
positions 77 to
70 bp and
49 to
42 bp, respectively. In
addition to the CREs, one putative Sp1 site (CCGCCC) is located at
position +42 to +47 bp relative to the major TSS. Other elements, such
as TREs, have not been characterized. In our study, deletional analysis
of hPC1 promoter constructs in transient transfection assay coupled
with EMSAs demonstrate that the region from
18 to +19 bp of the hPC1
promoter is likely responsible for the negative regulation by
T3. This region is near the TSS, similar to the observation
for nTREs described by others (40). Transcriptional repression by T3 can occur through several mechanisms,
including 1) binding of TRs to specific nTREs near TSS to
sterically interfere with components of transcription initiation
machinery (37) and 2) competitive binding of
TREs to other transactivation factors, in particular the Sp1
(52) and activator protein 1 (28) complex, and the estrogen receptor (41). Heterodimerization of
active proteins with inactive forms and competition for limiting
transactivation proteins may also occur (32, 47).
Moreover, complex interactions may involve more than one of these
principles on "composite" DNA response elements. The hPC1 promoter
lacks canonical TATA or CAAT boxes (19, 36). The potential
binding sites for transcriptional factors, such as Sp1, activating
transcription factor, and CRE binding protein are located beyond the
18 to +19 bp of hPC1 promoter. These findings suggest that nuclear
factors other than those listed above may be involved in T3
suppression of the hPC1 promoter.
RXR has been shown to heterodimerize with TR to increase TR/DNA
interactions (12). A number of studies have demonstrated that such heterodimerization can augment T3-mediated gene
regulation (9). However, the magnitude of the augmentation
varies significantly, especially when different cell lines are used. In
our study, both 9-cis-RA and T3 stimulated rGH
promoter, and the combination of T3 and 9-cis-RA
exerted additive effects on this promoter in GH3 cells, which is
consistent with the previous analysis (54). In contrast to
the rGH promoter, basal hTRH promoter luciferase activity was not
affected by 9-cis-RA, and the T3-mediated
inhibitory effect on the hTRH promoter was not altered by the
9-cis-RA. Interestingly, 9-cis-RA treatment also
increased hPC1 promoter activity and the addition of T3
reversed that stimulation to basal level, similar to our studies with
9-cis-RA and the hPC2 promoter (30). The effects on the hPC1 promoter could represent cumulative effects of
RXR-mediated stimulation and TR-mediated repression. Alternatively, TR
and RXR may competitively bind with the same DNA sequences located at
the 18 to +19 bp of hPC1. However, the lack of specific RXR binding
to the
18 to +19 bp of hPC1 promoter in the EMSA experiments (Fig.
7A) argues against these hypotheses. Moreover, when the
hPC1-971 promoter was transfected into JEG-3 cells (which express
high levels of endogenous RXR
), promoter activity was not
significantly increased by 9-cis-RA treatment in the
conditions with or without cotransfection of TR (data not shown). These
results suggest that other cell type-specific nuclear factors in
addition to TR/RXR may be required for either 9-cis-RA
and/or T3-mediated regulation.
Multiple TR isoforms are derived from two distinctive genes by
alternative promoter usage and alternative splicing of primary gene
products (27, 37). TR isoform specificity in regulation of
target gene expression has been demonstrated in myelin basic protein
genes (26) and in the rat TRH gene (29). In
the present study, we did not observe any significant functional
differences between TR1 and TR
1 isoforms in the hPC1 promoter in
either CV-1 or JEG-3 cells. These results were consistent with previous findings in human and mouse TRH genes (8, 40) and the hPC2 promoter (30). The lack of functional differences among
TR
1 and TR
1 isoforms in our study may be due to the different
nTREs in the different promoters and the levels of endogenous TR, RXR, and other cell type-specific transcriptional factors. Alternatively, the overexpression of TR in transient transfection assays may not
reflect the fine-tune regulation that occurs in vivo. In contrast to
some studies on T3 negatively regulated genes (8,
40), our study showed that cotransfection of either TR
1 or
TR
1 alone in both CV-1 and JEG-3 cells did not affect the basal PC1
promoter activity, but addition of T3 resulted in
suppression. These results were supported by the study of the nTRE of
hPC2 promoter (30) and epithelial growth factor receptor
promoter (52). We postulate that, taken together, some
untested or unidentified transcriptional factor may interact with both
TR and RXR to mediate T3 regulation on hPC1 promoter in a
manner similar to the hPC2 promoter.
Positive TREs generally are composed of paired hexameric half-sites
[(A/G)GGT(C/G)A] (27, 37). In addition, immediate flanking sequences of hexameric half-sites may also modulate TR-DNA interactions (50). The configuration of promoters
negatively regulated by thyroid hormone through nTREs is largely
unknown. Most nTREs identified so far, such as those in mouse and human TRH (29, 40), rat sodium, potassium ATPase
(48), TSH subunit and glycoprotein-
subunit
(5), exhibit variable half-site sequences, which may or
may not contain consensus TRE sequences. EMSAs using purified TR
1
and RXR proteins and GH3 nuclear extracts led us to further localize
the regions between the
10 and +19 bp of hPC1 promoter containing
putative nTREs. It is not surprising that this region is able to form a
TR
1/RXR heterodimer and multimer, because multiple TRE-like
hexameric sequences arranged presumably as direct repeat and inverted
palindrome are present (Fig. 6A). EMSAs with mutant
oligonucleotides, in which the core of two putative nTREs were mutated
(Fig. 7A), revealed diminished TR monomer and homodimer
binding (Fig. 7C, lanes 2 and 5). Most
interestingly, the mutant oligonucleotides still preserved TR/RXR
heterodimer but not multimer binding. Because of the diversity of TREs
identified in different genes, we hypothesize that the region between
10 and +19 bp of hPC1 promoter contains multiple TRE-like half-sites, which function in concert to mediate T3 regulation.
Transient transfection in GH3 cells supports this hypothesis, as the
construct with indicated point mutations exhibited a reduced, but not
abolished, effect of T3. This indicates that the negative
effects of T3 are likely mediated through a series of
partial nTREs in the region between
10 and +19 bp, and the mutated
nTREs partially contribute to the cumulative effect of T3.
Finally, in this paper, our in vivo results of rat anterior pituitary PC1 mRNA regulated by thyroid status confirms the findings of Day et al. (6) and are similar to the results of rat anterior pituitary PC2 mRNA regulated by thyroid status (30). Hypothyroidism induced a more profound stimulation of PC1 (this paper) than of PC2 mRNA (30) in the anterior pituitary. Additionally, T3 intraperitoneal injection for 3 days after prolonged PTU treatment decreased PC1 mRNA, whereas T3 administration (after PTU treatment) only returned PC2 mRNA to the control levels. These results indicate that PC1 mRNA is more susceptible to alterations of thyroid hormone than PC2 is. Examining the T3-responsive regions on the hPC1 and hPC2 promoter along with the in vivo observations, we hypothesize that the putative nTREs on the hPC1 promoter interact more strongly with TR/RXR and other nuclear proteins than the nTREs on the hPC2 promoter.
Thyroid hormones are essential for growth and development. Thyroid hormone also exerts specific effects on several organ systems, including cardiovascular, reproductive, central, and peripheral nervous systems (13). PC1 and PC2 process a wide variety of central and peripheral prohormones. In the anterior pituitary, PC1 and PC2 are found in corticotrophs, thyrotrophs, and gonadotrophs (49). In the corticotroph, PC1 and PC2 have clearly been shown to process POMC to ACTH (42). They may also process the precursors to substance P, neuropeptide Y, and vasoactive intestinal peptide, hormones found to be increased in the anterior pituitary of hypothyroid rodents (1, 22, 25). The simultaneous alterations of both PC1 and PC2 by thyroid hormone (compared with the alterations in either PC1 or PC2 alone in the patient deficient in PC1 and in mice with a knockout of PC2) may mediate more profound alterations in the levels of many hormones.
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ACKNOWLEDGEMENTS |
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This research was supported by Training Grant in Endocrinology and Diabetes DK-287235 (Q.-L. Li), Geconcenteerde Onderzoekacties 1997-2001 (E. Jansen), Veterans Affairs Medical Research Funds, and National Institutes of Health Grant DK-43714 (G. A. Brent), Thyroid Research Advisory Council (Knoll Pharmaceutical) Grant SYN-0400-02 (T. C. Friedman) and a National Institutes of Health Grant DA-00276 (T. C. Friedman). T. C. Friedman is also supported by Center of Clinical Research Excellence Grant (U54 RR-14616-01) to Charles R. Drew University of Medicine and Sciences and is a Culpeper Fellow.
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FOOTNOTES |
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Address for reprint requests and other correspondence: T. C. Friedman, Charles R. Drew Univ. of Medicine & Sciences, Division of Endocrinology, 1721 E. 120th St., Los Angeles, CA 90059 (E-mail: Friedmant{at}hotmail.com).
A portion of this work was presented at the 81st Annual Meeting of the Endocrine Society (1999).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 15 May 2000; accepted in final form 20 September 2000.
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