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Endocrinology Vol. 141, No. 9 3256-3266
Copyright © 2000 by The Endocrine Society

ARTICLES

Interactions between the Prohormone Convertase 2 Promoter and the Thyroid Hormone Receptor1

Qiao-Ling Li, Erik Jansen, Gregory A. Brent, Syed Naqvi, John F. Wilber and Theodore C. Friedman2

Division of Endocrinology, Department of Medicine, Cedars-Sinai Research Institute-University of California School of Medicine (Q.L.-L., S.N., T.C.F.), Los Angeles, California 90048; Laboratory for Molecular Oncology, Center for Human Genetics, University of Leuven and the Flanders Interuniversity Institute for Biotechnology (E.J.), B-3000 Leuven, Belgium; Division of Endocrinology, Department of Medicine, West Los Angeles Veterans Affairs Medical Center, and Departments of Medicine and Physiology, University of California School of Medicine (G.A.B.), Los Angeles, California 90073; and Division of Endocrinology and Metabolism, Department of Medicine, University of Maryland (J.F.W.), Baltimore, Maryland 21201

Address all correspondence and requests for reprints to: Theodore C. Friedman, M.D., Ph.D., Charles R. Drew University of Medicine and Science, Endocrinology Division, University of California, 1721 East 120th Street, Los Angeles, California 90059. E-mail: friedmant{at}hotmail.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The majority of prohormones are cleaved at paired basic residues to generate bioactive hormones by prohormone convertases (PCs). As PC1 and PC2, two neuroendocrine-specific PCs, appear to be the key enzymes capable of processing a variety of prohormones, alterations of PC2 and/or PC1 levels will probably have a profound effect on hormonal homeostasis. We investigated the regulation of PC2 messenger RNA (mRNA) by thyroid hormone using GH3 cells to demonstrate that T3 negatively regulated PC2 mRNA levels in a dose- and time-dependent fashion. Functional analysis of progressive 5'-deletions of the human (h) PC2 promoter luciferase constructs in GH3 cells demonstrated that the regulation probably occurs at the transcriptional level, and that putative negative thyroid hormone response elements were located within the region from -44 to +137 bp relative to the transcriptional start site. Transient transfections in JEG-3 cells and COS-1 cells showed that the suppressive effect of T3 was equally mediated by the thyroid hormone receptor (TR) isoforms TR{alpha}1 and TRß1. Electrophoretic mobility shift assays using purified TR{alpha}1 and retinoid X receptor-ß protein as well as GH3 nuclear extracts showed that regions from +51 to +71 bp and from +118 to +137 bp of the hPC2 promoter bind to TR{alpha}1 as both a monomer and a homodimer and with TR{alpha}1/retinoid X receptor-ß as a heterodimer. Finally, the in vivo regulation of pituitary PC2 mRNA by thyroid status was demonstrated in rats. These results demonstrate that T3 negatively regulates PC2 expression at the transcriptional level and that functional negative thyroid hormone response elements exist in the hPC2 promoter. We postulate that the alterations of PC2 activity may mediate some of the pathophysiological consequences of hypo- or hyperthyroidism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE MAJORITY OF prohormones are cleaved at paired basic residues to generate bioactive hormones in a cell-specific manner by prohormone convertases (PCs), members of the mammalian family of the subtilisin-like endoproteases (1, 2). Seven members of the PC family have been cloned: furin (3, 4), PC1 (2, 5, 6, 7) [also referred to as PC3 (1)], PC2 (1, 2), PACE4 (8), PC4 (9, 10), PC5/6 (11, 12), and PC7/PC8 (13, 14). Both PC1 and PC2 are found exclusively in neural and endocrine cells equipped with a regulatory secretory pathway (2, 5, 15, 16), PC4 is found in the testis (9), whereas the other PCs are ubiquitously distributed (17, 18). PC1 and PC2 process a variety of prohormones, including POMC, prosomatostatin, provasopressin, proneurotension, pro-TRH, and pro-CRH (19, 20, 21, 22, 23). The important roles of PC1 and PC2 in hormonal biosynthesis have been elucidated by the studies of mice lacking PC2 and in a patient with defective PC1. PC2 knockout mice have absent proglucagon processing, impaired proinsulin processing and were hypoglycemic (24, 25). The changes in pituitary hormones were not studied. A patient lacking PC1 had severe childhood-onset obesity, postprandial hypoglycemia, infertility, and low levels of ACTH and cortisol with elevated levels of POMC (26, 27).

In rat pituitary, PC1 is present in higher amounts in the anterior lobe, whereas higher levels of PC2 are found in the intermediate lobe (5, 16, 28). Rat intermediate lobe PC1, PC2, and POMC messenger RNA (mRNA) levels increased with treatment with the dopamine antagonist, haloperidol, and decreased with treatment with the dopamine agonist, bromocriptine (15, 21, 28). This regulation is at the level of transcription and involves the intracellular cAMP pathway (29). Regulation of processing enzymes by thyroid status has been briefly studied in rat pituitary. Hypothyroid rats induced by thyroidectomy or 6-n-propyl-2-thiouracil (PTU) treatment had an increase in anterior pituitary PC1, PC2 (15), and peptidylglycine {alpha}-amidating monooxygenase (a processing enzyme involved in amidation) mRNA levels (30) and a decrease in PACE4 (a more ubiquitously distributed PC) mRNA levels (31). Rats made hyperthyroid by daily injection of T4 showed decreased anterior pituitary PC1, PC2, and peptidylglycine {alpha}-amidating monooxygenase mRNA levels and increased PACE4 mRNA levels (15, 31).

In many well studied examples, such as in the regulation of TRH, TSH, GH, and {alpha}-myosin heavy chain, thyroid hormone has been shown to act through nuclear thyroid hormone receptors (TRs) at the transcriptional level (32, 33, 34, 35, 36). It is not yet known whether the effects of thyroid hormone on PC1 and/or PC2 mRNA expression observed in rat anterior pituitary are mediated directly through TR and whether PC1 and PC2 promoters contain negative thyroid hormone response elements TREs (nTREs).

Thyroid hormone (T3) regulates target gene expression by binding with TRs. T3/TR mediate transcriptional regulation through interactions in the promoter region of target genes with consensus DNA sequences, referred to as the thyroid hormone response element (TRE) (37). Positive TREs generally are composed of hexameric half-sites [(A/G)GGT(C/G)A] arranged as direct repeats, palindromes, or inverted palindromes (37, 38). In addition, immediate flanking sequences of hexameric half-sites may modulate TR-DNA interaction (39, 40). The configurations of promoters negatively regulated by thyroid hormone through nTREs are largely unknown. Most of the nTREs identified to date, such as those in mouse and human TRH (41, 42, 43), rat sodium/potassium-adenosine triphosphatase (44), TSH ß-subunit, and glycoprotein {alpha}-subunit (45, 46), exhibit variable half-site consensus sequences. Moreover, in both T3-positive and -negative regulation models, heterodimerization between TR and retinoid X receptor (RXR) augments the ligand-dependent stimulation or repression (37, 47). However, in some unique TRE-containing promoters, such as that in human type 1 deiodinase promoter, RXR-independent mechanisms are involved in thyroid hormone regulation (40).

To further address the mechanism of T3 regulation of the PC2 gene, we used GH3 cells, a rat sommatotrope cell line expressing endogenous TRs (48) and PC2 (18, 49), to study the regulation of PC2 mRNA levels by T3. We used transient transfection assays with progressively truncated human (h) PC2 promoter luciferase constructs to localize the region of T3 regulation in the hPC2 promoter. Electrophoretic mobility shift assays (EMSAs) were performed using oligonucleotides selected from -44 to +137 bp of the hPC2 promoter to further localize putative nTREs. Finally, the in vivo regulation of pituitary PC2 mRNA by thyroid status was confirmed in rats by Northern blots. We hypothesize that alterations of PC2 activity by thyroid hormone may mediate some of the pathophysiological consequences of hypo- or hyperthyroidism.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
GH3 cells (rat sommatotrope cells) and JEG-3 cells (human choriocarcinoma cells) were obtained from American Type Culture Collection (Manassas, VA) and maintained at 37 C and 5% CO2 in DMEM with 10% FBS (Life Technologies, Inc., Gaithersburg, MD) supplemented with 0.075% sodium bicarbonate, 50 IU/ml penicillin-streptomycin, and 0.125 mg/ml fungizone (Amphotericin, Life Technologies, Inc.). CV-1 (African green monkey kidney cells) cells were provided by Dr. Philip Koeffler (Cedars-Sinai Medical Center, Los Angeles, CA) and were grown in MEM supplemented with 10% FBS, 2 mM L-glutamine, 0.11 mg/ml sodium pyruvate, and nonessential amino acids.

Northern blot analysis of PC2 mRNA in GH3 cells
GH3 cells were seeded in 100-mm tissue culture dishes and allowed to adhere overnight. Cells were washed with PBS and serum starved for 16 h to synchronize the cell cycle. The cells were subsequently incubated in serum-free medium supplemented with 5% charcoal-stripped FBS in the presence of T3 (10-8 M) for 6–48 h or in the presence of different doses of T3 (10-10–10-7 M) for 24 h. The addition of T3 was staggered so that all of the cells (including the control cells that were not treated with T3) were harvested simultaneously. Total RNA (20 µg), extracted from GH3 cells using TRIzol reagent (Life Technologies, Inc.), was loaded onto a 1.2% formaldehyde denaturing agarose gel. After horizontal electrophoresis, the gel was transferred to a GeneScreen Plus hybridization transfer membrane (NEN Life Science Products, Boston, MA). The pBluescript-rPC2 construct, provided by Dr. Richard Mains (The Johns Hopkins University, Baltimore, MD), was used for generating the PC2 riboprobe. A 531-bp [32P]UTP-labeled rPC2 antisense riboprobe was synthesized using T7 RNA polymerase (Life Technologies, Inc.). The membrane was hybridized with the probe at 60 C in 50% formamide, 5 x SSC (standard saline citrate), 1% SDS, 5 x Denhardt’s, 5% dextran sulfate, and 100 µg/ml salmon sperm DNA and washed at room temperature with 2 x SSC-0.1% SDS, followed by two washes at 70 C for 1 h with 0.1 x SSC-0.1% SDS. The blot was developed by exposure to x-ray film with an intensifying screen for 16–18 h, then stripped with Probe Degradation Buffer (Ambion, Inc., Austin, TX) and Blot Reconstitution Buffer (Ambion, Inc.) and further probed with an 111-bp rat cyclophilin riboprobe (50) using the same conditions as those described above. The mRNA for PC2 was quantitated using the NIH Image 1.61 program, with cyclophilin as an internal control. The intensity of the signal was linear with the amount of RNA.

Plasmid constructs and luciferase assays
The hPC2 luciferase fusion gene expression regions of the plasmids were constructed by subcloning progressively truncated hPC2 promoter with 5'-ends at -4.5 kb, -789 bp, -226 bp, -110 bp, -84 bp, and -44 bp relative to the transcription start site (TSS) into the polylinker region of the promoterless, luciferase-encoding pGL2-basic plasmid (Promega Corp., Madison, WI). The 3'-end of all constructs ended at position +137 bp relative to the TSS. The human TRH (hTRH) luciferase construct was generated by subcloning -900 to +54 bp relative to TSS of hTRH promoter into the promoterless pA3-luciferase plasmid (51). The rat GH (rGH) luciferase construct was generated by subcloning the fragment from -528 to +65 bp relative to TSS of rGH promoter into the promoterless pA3-luciferase plasmid. The TK-105 luciferase construct was provided by Dr. Lin Pei (Cedars-Sinai Medical Center).

GH3 cells were plated in growth medium in six-well plates and allowed to adhere overnight. Cells were then placed in Opti-MEM (Life Technologies, Inc.) medium and overlaid with a mixture of DNA/cationic liposomes (Lipofectamine, Life Technologies, Inc.). Cells were incubated with DNA (3 µg/well) for 6 h, the medium was changed to DMEM serum-free medium for overnight incubation, and cells were treated with either T3 (10-8 M), 9-cis-retinoic acid (9-cis-RA; 10-7 M), or their combination for 16 h. Cells were then lysed in 25 mM Tris-phosphate buffer (pH 7.8), 10 mM MgCl2, 0.1% BSA, 15% glycerol, 1% Triton X-100, and 1 mM EDTA. After centrifugation, 180 µl of the cleared cell lysate were used for the luciferase assay. Luciferase activity was measured in a Berthold Lumat LB 9501 luminometer (Wallac, Inc., Gaithersburg, MD) in the presence of 0.8 mM ATP and 0.3 mM D-luciferin. Integrated light emission over 15 sec was measured. All transfections were performed in triplicate and repeated at least three times.

To study the effects of different isoforms of TR mediating the T3 regulation of PC2, JEG-3 and CV-1 cells were seeded overnight into 60-mm dishes, and the medium was changed 2–3 h before transfection by calcium phosphate precipitation. Three micrograms of hPC2 promoter-luciferase constructs, 0.5 µg of the different isoforms of TR (TR{alpha}1, TR{alpha}1, and TRß1; TRß2 was not used because of poor transfection efficiency using this plasmid), and 0.5 µg carrier plasmid were used for transfection. One microgram of the RSV0-ß-galactosidase plasmid (ß-gal) was used for measuring the transfection efficiency (32). Six hours after transfection, the cells were incubated in the presence or absence of T3 (10-8 M) for 24 h. All experiments were performed in duplicate plates, and each experiment was repeated three to five times.

Nuclear extract preparation and electrophoretic mobility shift assays (EMSAs)
Nuclear extracts were prepared from GH3 cells according to the method of Dignam et al. (52). Protein concentrations in nuclear extracts were determined with the Bio-Rad Laboratories, Inc. protein assay kit (Hercules, CA). The region of the hPC2 promoter from -44 to +137 bp relative to the TSS was generated by PCR. Double stranded oligonucleotides corresponding to the human PC2 gene promoter DNA sequence -20 to +15 bp (O#1), +29 to +47 bp (O#2), +51 to +71 (O#3), and +118 to +137 bp (O#4) relative to the TSS were custom synthesized by Life Technologies, Inc., and radiolabeled using [{gamma}-32P]ATP (6000 Ci/mmol; NEN Life Science Products, Boston, MA) and T4 polynucleotide kinase (Life Technologies, Inc.). EMSAs were performed as follows. GH3 nuclear proteins (0.5 µg), purified TR{alpha}1 (0.01–0.1 µg; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or RXRß (2.0 µg; BIOMOL Research Laboratories, Inc., Plymouth Meeting, PA) proteins were incubated for 10 min 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 20 µg/ml poly(dI-dC) as a nonspecific competitor. Subsequently, radiolabeled DNA probes were added, and the incubation was continued for another 20 min. For competition and antibody supershift experiments, binding mixtures were incubated with unlabeled double stranded oligonucleotides or specific antibodies for 10 min before addition of the radiolabeled oligonucleotides. All specific antibodies were obtained from Santa Cruz Biotechnology, Inc.. Protein-DNA complexes were analyzed on 5% nondenaturing polyacrylamide gels at 25 C in 0.5 x Tris borate/EDTA buffer and visualized by autoradiography.

Northern blot analysis of pituitary from rats receiving PTU and T3
Adult male Sprague Dawley rats, weighing around 250 g, were housed in a light- and temperature-controlled environment and fed standard laboratory rat chow. Hypothyroidism was induced in rats by adding PTU (0.05%) to their drinking water for 2 weeks. Some hypothyroid rats then received daily ip injection of T3 (300 µg/kg) for 3 days; the rest of animals received daily ip injection of vehicle for 3 days before death. Animals were then euthanized by CO2 administration, and the pituitaries were immediately collected and separated into anterior and neurointermediate lobes. Total RNA from individual pituitary lobes was extracted using TRIzol. Five micrograms of total RNA were fractionated on 1.2% denaturing agarose gel. Northern blots were carried out as described above. The protocol was conducted using NIH guidelines and was approved by the Institutional Animal Care and Use Committee of Cedars-Sinai Medical Center.

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 the Dunnett correction factor) for comparing treatment with control.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Time-course and dose-response regulation of PC2 mRNA by T3 in GH3 cells
We used GH3 cells, which are frequently used to study T3 regulation of gene expression (33), to study the effect of thyroid hormone on PC2 gene expression. GH3 cells treated with a range of doses of T3 (10-7–10-10 M) for 24 h (Fig. 1Go, A and B) exhibited a significant down-regulation (2.0- to 2.8-fold) of PC2 mRNA in a dose-dependent fashion (F = 18.10; P < 0.0001), which was maximal at 10-8 M. Moreover, GH3 cells treated with T3 (10-8 M) for 6–48 h (Fig. 1Go, C and D) showed a significant time-dependent reduction in PC2 mRNA (F = 12.86; P = 0.0006), beginning at 6 h and maximal at 24 h (1.9- to 3.9-fold). These results indicate that T3 regulates PC2 mRNA in a dose- and time-dependent manner consistent with transcriptional regulation.



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  		Figure 1. Time course and dose response of T3 negative regulation of PC2 mRNA in GH3 cells. A, Representative Northern blot analysis of PC2 mRNA in GH3 cells treated with 10-7-10-10 M of T3 for 24 h. B, Quantitation of dose response of Northern blots expressed as the mean ± SEM corrected to the control (without T3) from three independent experiments. C, Representative Northern blot analysis of PC2 mRNA in GH3 cells treated with T3 for 6–48 h (10-8 M). D, Quantitation of time course of Northern blots expressed as the mean ± SEM corrected to the control value (without T3) from three independent experiments. *, P < 0.01 (compared with the control).

 
Regulation of hPC2 promoter by T3 and 9-cis-RA
After demonstrating that T3 negatively regulates endogenous PC2 mRNA in GH3 cells, we examined the effects of T3 alone and in combination with 9-cis-RA on expression of the hPC2 promoter in GH3 cells. We used the hPC2–789 promoter luciferase construct, which has been described in previous studies (53). The thymidine kinase (TK)-105 construct, the hTRH promoter from -900 to +54 bp (32), and the rGH promoter from -528 to +65 bp, all cloned in front of luciferase reporter gene, were used as T3-responsive controls for transfection in GH3 cells. As shown in Fig. 2Go, T3 (10-8 M) inhibited hPC2 promoter activity by 45% (P < 0.05), whereas 9-cis-RA (10-7 M) alone increased (2.7-fold; P < 0.001) hPC2 promoter luciferase activity. Addition of T3 reversed the 9-cis-RA stimulation to the basal level (P = NS compared with control; P < 0.05 compared with 9-cis-RA treatment). Consistent with previous observations (54, 55), T3 and 9-cis-RA treatment resulted in 2.5- and 3-fold stimulations of rGH promoter. The combination of 9-cis-RA and T3 resulted in an additive effect. In contrast to the hPC2 promoter, the hTRH promoter was not affected by 9-cis-RA alone (P = NS), and the T3-mediated down-regulation of hTRH promoter was not affected by the addition of 9-cis-RA in GH3 cells. Furthermore, the TK promoter was not affected by 9-cis-RA, T3, or their combination. Taken together, these results suggest the 9-cis-RA-positive regulation and T3-negative regulation of hPC2 promoter are unique.



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  		Figure 2. Effect of T3 and 9-cis-RA on hPC2 promoter activity. GH3 cells were transfected with the hPC2-luciferase, rGH-luciferase, hTRH-luciferase, and TK-luciferase constructs using the DNA cationic/liposomes method. After transfection and serum starvation, the cells were treated with T3 (10-8 M), 9-cis-RA (10-7 M), or their combination for 16 h. Cells were then harvested for luciferase assay as described. The results are expressed as the mean ± SEM of luciferase activity from three independent experiments. *, P < 0.05; **, P < 0.001 (compared with the control).

 
Deletion analysis of the hPC2 promoter in GH3 cells
To further analyze T3-mediated transcriptional regulation and characterize the putative TREs within the hPC2 promoter, we performed a series of transient transfection assays in GH3 cells with progressive 5'-deletions of hPC2 promoter luciferase reporter constructs. The results are expressed as percentage of repression compared with control cells (without T3). As shown in Fig. 3Go, the constructs containing hPC2 promoter DNA from -4.5 kb to +137 bp and from -789 to +137 bp relative to the TSS revealed similar T3-mediated suppression, which was 45–60% of the control value (without T3). Sequential deletion of -789 to -44 bp relative to TSS did not abolish T3-mediated down-regulation of luciferase activity. These results suggest that the nTRE(s) is likely to be located near the TSS, and the sequence between -44 to +137 bp of hPC2 promoter is sufficient to mediate the T3 inhibitory effect of hPC2 promoter. Similar inhibition by T3 was obtained using the hTRH promoter (Fig. 3Go). In contrast, T3 caused a 2.5-fold stimulation of rGH promoter (56). No influence of T3 on the TK promoter or pGL2 basic luciferase activity was observed, and the activity of pGL2 basic plasmid was minimal. However, unlike rGH and hTRH promoters, no consensus TRE elements were identified by computer homology match with published nTRE elements between the region from -44 to +137 bp of the hPC2 promoter.



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  		Figure 3. Deletion analysis to localize the putative TREs in hPC2 promoter. Different hPC2 promoter constructs were transfected into GH3 cells, incubated in the presence or absence of T3 (10-8 M) for 16 h, and then harvested for luciferase assay. The basal promoter activity of the -44 to +137 bp construct was approximately 30% of the activity of the -789 to +137 bp construct (the construct with the greatest basal promoter activity). The results are expressed as the mean ± SEM of luciferase activity compared with the control (without T3) from three independent experiments. *, P < 0.05; **, P < 0.001 (compared with the control).

 
TR isoform on the T3-repressive effect of hPC2 promoter in JEG-3 cells and CV-1 cells
To study whether T3/TR-mediated repression of hPC2 promoter activity in GH3 cells is TR isoform specific, we performed cotransfection experiments (TR isoforms and the hPC2 promoter) using JEG-3 and CV-1 cells, which are known to be relatively deficient of endogenous TRs (41, 57). Despite the fact that JEG-3 and CV-1 cells do not express PC2, high levels of PC2 promoter luciferase activity were found when the PC2 promoter construct was transfected into these cells. This finding is similar to the high luciferase activity obtained when the hTRH promoter construct was transfected into JEG-3 and CV-1 cells (43). As shown in Fig. 4Go, T3 (10-8 M) alone did not influence basal PC2 promoter luciferase activity in the two cell lines tested. However, unlike the results for human TRH (32) and mouse TRH (41) promoters, cotransfection of TR isoforms (TR{alpha}1, TR{alpha}2, or TRß1) alone did not increase basal hPC2 promoter activity. Addition of T3 to the cells cotransfected with either TR{alpha}1 or TRß1 resulted in a 55–75% reduction in luciferase activity, in both cell lines. In contrast, addition of T3 in the cells cotransfected with TR{alpha}2 alone, the TR isoform that does not bind T3 (37), did not affect hPC2 promoter activity, as anticipated. These results indicate that T3-mediated PC2 gene expression is dependent on the presence of functional TRs, and that there is no TR{alpha}1 or TRß1 isoform preference in the regulation of hPC2 promoter in the transient transfection system used in our study.



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  		Figure 4. TR isoform effect on the repressive effect of T3 on hPC2 promoter activity in CV-1 cells and JEG-3 cells. CV-1 and JEG-3 cells were transfected with -789/+137 bp hPC2 promoter construct with different TR isoforms and were incubated in the presence or absence of 10-8 M T3 for 24 h after transfection and were harvested for luciferase assay. The results are expressed as the mean ± SEM of luciferase activity compared with the control (without T3). *, P < 0.05 (compared with the control).

 
Binding of purified TR{alpha}1 and RXRß proteins to the hPC2 -44 to +137 bp fragment in EMSAs
We next determined whether TR binds to the functionally defined fragment from -44 to +137 bp of the hPC2 promoter. EMSAs were carried out using purified recombinant TR{alpha}1 and RXRß proteins. The probes were the hPC2 promoter (from -44 to +137 bp) and the DR4 element, a 16-nucleotide synthetic oligonucleotide that contains 2 consensus TREs (AGGTCA) separated by 4 nucleotides, which was used as a positive control (58). As shown in Fig. 5Go, 2Go bands corresponding to TR monomer (M) and homodimer (HOD) were observed using either the DR4 (Fig. 5AGo) or PC2 (Fig. 5BGo) fragments in those binding reactions containing only purified TR{alpha}1 protein (lanes 1–5). The binding to both DR4 and hPC2 was decreased when the concentration of TR{alpha}1 was decreased from 0.1 µg (lanes 1–4) to 0.01 µg (lanes 5–9) in the reactions. The intensity of the bands was decreased by competition with a 100-fold molar excess of unlabeled identical oligonucleotides (lane 3), but not by a 200-fold molar excess of nonspecific competitor (lane 4). RXR alone, at a concentration approximately 10-fold higher than the highest concentration of TR{alpha}1 (lane 2), did not show any binding to either DR4 or hPC2 (lane 6) fragments. Because an optimal ratio of TR and RXR proteins in the reaction is required for efficient TR/RXR heterodimer formation, we chose a small amount of TR{alpha}1 (0.01 µg) to show the interaction between TR and RXR on both DR4 and hPC2 fragments. When both TR{alpha}1 and RXRß were added, a strong TR/RXR heterodimer band (HED) was detected for DR4 and hPC2 (lane 7). This band has slightly faster mobility than TR homodimer due to the smaller molecular mass of purified RXR (51 kDa) compared with that of the TR (73 kDa) protein. Interestingly, a large-sized oligomer band (Oligo) of TR/RXR was also observed using the hPC2 fragment (Fig. 5BGo, lane 6). The binding of the oligomer, and to a lesser extent of the heterodimer, was partially inhibited by the presence of anti-TR and anti-RXR antibody for both DR4 and hPC2 (lanes 7 and 8), confirming the binding specificity.



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  		Figure 5. The binding of -44/+137 bp hPC2 promoter fragment to purified TR{alpha}1 and RXRß. EMSAs were performed using double stranded DR4 element (A) corresponding to direct repeat of consensus half-site AGGTCA separated by four nucleotides and the -44/+137 bp hPC2 fragment synthesized by PCR (B) The monomer (M), homodimer (HOD), heterodimer (HED), and oligomer (Oligo) bindings are indicated by arrows. NON, Nonspecific competitor.

 
EMSAs using four oligonucleotides selected from -44 to +137 bp of the hPC2 promoter
By computer homology search, we were not able to identify any consensus TRE sites within -44 to +137 bp of the hPC2 promoter. However, four oligonucleotides containing partial TRE-like sequences were selected (Fig. 6AGo), -20/+15 bp (O#1), +29/+47 bp (O#2), +51/+71 bp (O#3), and +118/+137 bp (O#4), with the numbers referring to the nucleotides compared with the TSS. EMSAs were performed with these probes using increasing amounts of purified TR{alpha}1. As indicated in Fig. 6BGo, no binding was detected using O#1, which contains the major TSS within the hPC2 promoter. Interestingly, both monomer and homodimer binding were observed using O#3 and O#4 with increasing intensity of binding when increasing amounts of TR{alpha}1 protein were included in the reactions. In contrast, O#2 bound to TR{alpha}1 protein with much weaker binding affinity than O#3 and O#4; faint mono- and homodimer binding was observed only with the higher amounts of TR{alpha}1 protein added to the reactions. In preliminary experiments, we also found that O#3 and O#4 were more effective than O#1 and O#2 at competing for the binding of DR4 to GH3 cell nuclear extracts (data not shown). Taken together, these results suggest that the regions between +51 to +71 bp and +118 to +137 bp on hPC2 promoter are capable of binding to TR with relative high binding affinity.



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  		Figure 6. The binding of four oligonucleotides selected from -44 to +137 bp of the hPC2 promoter to purified TR{alpha}1 and RXRß and the ability of those oligonucleotides to compete with labeled DR4 for binding to GH3 nuclear extracts. A, Sequences of four oligonucleotides selected from -44 to +137 bp of hPC2 promoter. Arrows indicate the putative TRE half-sites containing at least four of the six nucleotides of the consensus TRE [(A/G)GGT(C/G)A]. B, Binding of the four double stranded oligonucleotides probes selected from the hPC2 promoter to increasing amounts of TR{alpha}1 protein (0, 0.01, 0.04, and 1.0 µg).

 
EMSAs using O#3 and O#4 with TR{alpha}1 and RXRß proteins
As we observed no binding using O#1, very weak binding using O#2 with TR{alpha}1, and no augmentation of RXR (data not shown) using these two oligonucleotides, we chose O#3 and O#4 to further test TR/RXR interaction in these two regions, We speculated that these two regions may contribute to the multiple oligomers observed using the -44 to +137 bp fragment (Fig. 5BGo). EMSAs were carried out using purified TR{alpha}1 and RXRß proteins with O#3 and O#4 fragments as probes. As indicated in Fig. 7Go, when higher amounts of TR{alpha}1 were added to the reactions, both TR monomer and homodimer were detected (lane 2) with O#3 and O#4, as anticipated. The addition of T3 diminished the homodimer binding (lane 3), but had no effect on monomer binding, which is consistent with observations of other consensus TRE sequences (59, 60). TR{alpha}1 antibody diminished the binding, suggesting the specificity of the interaction. In those lanes with a low (0.01 µg) amount of TR{alpha}1 added to the reactions, faint TR homodimer with a larger amount of monomer binding was detected (lane 5). RXR itself did not bind to either O#3 or O#4 (lane 6). Most interestingly, intense TR/RXR heterodimer was detected with both probes (lane 7), which was more intense with O#3 (Fig. 7AGo, lane 7). T3 increased the intensity of heterodimer binding of O#3 (Fig. 7AGo, lane 8). The addition of TR antibody (lane 9) inhibited the binding of both probes. These results indicate that not only the TR monomer and homodimer, but also TR/RXR heterodimer, can be formed with both the O#3 and O#4 probes.



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  		Figure 7. The binding of two oligonucleotides (O#3 and O#4) to TR{alpha}1 and RXRß proteins. Two oligonucleotides from +51 to +71 bp (O#3, A) and from +118 to +137 bp (O#4, B) of the hPC2 promoter were used as probes for EMSAs. The monomer (M), homodimer (HOD), and heterodimer (HED) bindings are indicated by arrows. Lane 1, Probe alone; lane 2, probe and 0.1 µg of TR{alpha}1; lane 3, probe, TR{alpha}1 and T3 (10-6 M); lane 4, probe, TR{alpha}1 and anti-TR{alpha}1 antibody; lane 5, probe and 0.01 µg TR{alpha}1; lane 6, probe and RXRß alone; lane 7, probe, TR{alpha}1 and RXRß; lane 8, probe, TR{alpha}1, RXRß and T3 (10-6 M); lane 9, probe, TR{alpha}1, RXRß, and anti-TR{alpha}1 antibody.

 
Regulation of pituitary PC2 mRNA in vivo by thyroid status
Thyroid hormone regulates many genes in the anterior pituitary. Regulation of PC1 and PC2 mRNA in anterior pituitary by thyroid hormone has also been demonstrated previously by one group (15). However, no further studies were conducted. To confirm the previous findings and establish the physiological relevance of our observation on the regulation of hPC2 by T3, Sprague Dawley rats were made hypothyroid by adding PTU to the drinking water for 2 weeks. In one group of rats, thyroid hormone levels were acutely elevated by ip injection of T3 3 days before the animals were killed. The hypothyroidism of PTU-treated rats was confirmed by increased TSH levels (mean ± SEM, 28.2 ± 3.3 mU/liter; P < 0.0001 compared with control) compared with the TSH levels in control rats (2.8 ± 1.0 mU/liter). The effectiveness of T3 treatment was confirmed by suppressed TSH levels (0.20 ± 0.03 mU/liter; P < 0.0001 compared with PTU-treated rats). The body weight of PTU-treated rats was significantly lower than that of control animals (data not shown). As shown in Fig. 8Go, A and B, administration of PTU resulted in a significant increase (2-fold) in PC2 mRNA levels in the anterior pituitary. T3 supplement after PTU treatment for 3 days significantly restored PC2 mRNA to the control level, but did not further lower PC2 levels compared with the control values. In agreement with the previous findings (15), no changes in PC2 mRNA in the neurointermediate lobe were observed with PTU or PTU/T3 treatment (Fig. 8Go, C and D).



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  		Figure 8. Northern blot analysis of pituitary PC2 mRNA from rats receiving PTU and T3. Representative Northern blot analysis of PC2 mRNA in anterior lobe (A) and neurointermediate lobe (B) of the pituitary from individual rats receiving PTU and T3 treatment. Northern blot analysis of cyclophilin mRNA and ethidium bromide staining of the 28S RNA band are also depicted. The quantitative results of PC2/cyclophilin mRNA levels from three animals per treatment group are expressed as the mean ± SEM induction over the control value (C) for anterior lobe and for neurointermediate lobe of the pituitary (D). *, P < 0.05 compared with the control; **, P < 0.05 compared with the group with PTU treatment.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The effects of thyroid hormone are protean. In the pituitary, thyroid hormone directly or indirectly regulates gene expression in all cell types (61, 62). The prohormone convertases, including PC2, are important processing enzymes that determine the ratio of many inactive prohormone precursors to active hormones (63). In this study we present evidence demonstrating that the T3 negative regulation of PC2 mRNA observed in vivo in rat anterior pituitary is at least in part due to direct T3 interaction with TR on nTREs in the hPC2 promoter.

Regulation of PC1 and/or PC2 mRNA by intracellular PKC and PKA pathways has been demonstrated (29, 53, 64, 65, 66). The promoter region of human PC2 contains one cAMP response element-like sequence (TCACGTCA) at position -53 to -46 bp relative to TSS, which is essential for neuroendocrine specificity of PC2 gene expression (53). In addition to the cAMP response element, the transcription factor early growth response gene-1 (EGR-1) has been found to interact with two elements on the hPC2 promoter (53). Others elements, such as TREs, have not been characterized. In our study truncational analysis of the hPC2 promoter luciferase reporter constructs in transient transfection assays demonstrated that the region from -44 to +137 bp of hPC2 promoter contains T3 regulation elements. This region is near the TSS, similar to the observation for nTREs described by others (41, 43). Transcriptional repression by T3 can occur through several mechanisms, including binding of TRs to specific nTREs near TSS to sterically interfere with TATA box and/or transcriptional initiation machinery (38) as well as competitive binding of TREs to other trans-activation factors, such as Sp1 (67), activating protein-1 (68), and the estrogen receptor (69, 70). Heterodimerization of active proteins with inactive forms and competition for limiting trans-activation proteins may also occur (32, 71, 72). Moreover, complex interactions may involve more than one of these principles on composite DNA response elements. The hPC2 promoter lacks canonical TATA or CAAT boxes (53, 73). The potential binding sites for transcriptional factors, such as Sp1, CAAT box transcription factor, EGR-1, and activating transcription factor/cAMP response element-binding protein, are located 5' compared with the region of -44 to +137 bp of hPC2 promoter (53, 73), the region shown in this study responsible for the T3-negative regulation. Moreover, the oligonucleotide (designed O#1) selected from hPC2 promoter adjacent to TSS and near well characterized transcription factors did not show any binding to purified TR{alpha}1 or RXR (data not shown), suggesting that other nuclear factors may be involved in T3 suppression of the hPC2 promoter.

RXR has been shown to heterodimerize with TR to increase TR/DNA interactions (58, 74). A number of studies have demonstrated that such heterodimerization can augment T3-mediated gene regulation (47, 60). However, the magnitude of the augmentation varies significantly, especially when different cell lines were 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 in GH3 cells, consistent with the previous analysis (75). In contrast to the rGH promoter, basal hTRH promoter luciferase activity was not affected by 9-cis-RA treatment, and the T3-mediated inhibitory effect on the hTRH promoter was not augmented by 9-cis-RA treatment in GH3 cells. Interestingly, 9-cis-RA treatment resulted in markedly increased hPC2 promoter activity; the addition of T3 reversed this stimulation to the basal level. This result probably represents cumulative effects of RXR-mediated stimulation and TR-mediated repression of hPC2 promoter. Alternatively, TR and RXR may competitively bind with the same consensus DNA sequence located at the -44 to +137 bp region of the hPC2 promoter. However, the lack of specific RXR binding to -44 to +137 bp of hPC2 promoter in the EMSA experiments argues against this hypothesis. Moreover, hPC2–789 promoter activity, when transfected into JEG-3 cells, which has been shown to express high levels of endogenous RXRß, was not significantly increased by 9-cis-RA treatment in experiments with or without cotransfection of TR (data not shown). These results suggest that other cell type-specific nuclear factors in addition to RXR may be involved in both 9-cis-RA- and T3-mediated regulation.

Multiple TR isoforms are derived from two distinctive genes by alternative promoter usage and alternative splicing of primary gene products (37, 38). Expressions of TR isoforms are regulated developmentally in different tissues (76, 77). TR isoform specificity in regulation of target gene expression has been demonstrated in myelin basic protein genes (78, 79). In the present study we did not observe any significant functional differences among TR{alpha}1 and TRß1 isoforms in the hPC2 promoter in either CV-1 or JEG-3 cells. These results are consistent with the previous findings in the T3-negative regulation of human and mouse TRH genes (41, 80), but are in contradiction to those studies with the rat TRH gene (42). The discrepancy of these results is most likely due to the different nTREs in the different promoters and the concentrations of endogenous TR, RXR, and other cell type-specific transcriptional factors. Alternatively, the overexpression of TR in transient transfection assays may not reflect the finely tuned regulation that occurs in vivo. In contrast to some studies on T3 negatively regulated genes (41, 80), our study showed that cotransfection of either TR{alpha}1 or TRß1 alone in both CV-1 and JEG-3 cells did not affect the basal PC2 promoter activity, but addition of T3 resulted in suppression. However, our results are supported by the study of the nTRE of epidermal growth factor receptor promoter (67, 81), in which TR alone did not effect promoter activity, indicating that the binding of TR to the TRE did not direct trans-activation. Moreover, the inhibitory effect of T3 on epidermal growth factor receptor was seen only as inhibition of Sp1-stimulated transcription. In the hPC2 promoter, some untested or unidentified transcriptional factor may interact with both TR and RXR to mediate T3 regulation, as discussed above.

EMSAs using purified TR{alpha}1 and RXRß proteins led us to localize two regions at +51 to +71 bp (O#3) and +118 to +137 bp (O#4) of the hPC2 promoter. Although no 5'-AGGTCA-3' consensus sequences were present in these two regions, there is an imperfect (five of six nucleotides) single half-site arranged in the 3' to 5' direction for both O#3 and O#4. This is especially interesting, as O#1 had several imperfect half-sites, and O#2 had an imperfect (five of six nucleotides) half-site arranged in the 5' to 3' direction, but both failed to bind to purified TR{alpha}1, RXRß, or their combination (data not shown). It is not yet known whether the orientation of the single half-site is critical for nTREs, which, in general, have more variability than positive TREs. Although most half-site nTREs are hexamers in the 5' to 3' direction, a functional half-site nTRE hexamer in the 3' to 5' direction has also been reported (82). It is noteworthy that although only a single half-site is found in O#3 and O#4 (Fig. 6AGo), both probes are able to form monomer and homodimer binding with TR and heterodimer binding with RXR. It is possible that in addition to our partial TREs outlined in Fig. 6AGo, there are additional nucleotides adjacent to the putative TREs that direct TR/RXR heterodimer formation.

Finally, in this paper our in vivo results of rat pituitary PC2 mRNA regulation by thyroid status confirm the findings of Day et al. (15). PTU treatment elevated PC2 mRNA levels, and T3 treatment for 3 days after PTU treatment restored PC2 levels to the control values. However, we did not detect the further reduction of PC2 mRNA in those animals receiving T3 injection (for 3 days), whereas TSH levels were suppressed by treatment with T3. Thus, PC2 down-regulation may require a longer period of hyperthyroidism than TSH down-regulation. We have recently demonstrated that PTU- induced hypothyroidism stimulated, whereas T3-induced hyperthyroidism suppressed PC1 mRNA in rat anterior pituitary (83). The simultaneous alterations of both PC1 and PC2 by thyroid hormone may mediate profound alterations in prohormone processing.

Thyroid hormone is essential for growth and development. Thyroid hormone also exerts specific effects on several organ systems, including cardiovascular, reproductive, and central and peripheral nervous systems (84). PC2 processes a wide variety of central and peripheral prohormones. We speculate that alterations of PC2 activity by thyroid hormone may mediate some of the pathophysiological consequences of hypo- or hyperthyroidism. This is corroborated by the finding that increased anterior pituitary levels of substance P, neuropeptide Y, and vasoactive intestinal peptide are found in hypothyroid rodents (85, 86, 87). Although the elevated levels of these hormones may be due to regulation of gene expression, they may also be due to altered prohormone processing.


   Footnotes
 
1 This work was supported by Training Grant in Endocrinology and Diabetes DK-287235 (to Q.-L.L.), Geconcentreerde Onderzoekacties 1997–2001 (to E.J.), V.A. Medical Research Funds and NIH Grant DK-43714 (to G.A.B.), and NIH Grant DA-00276 (to T.C.F.). Back

2 Charles. E. Culpeper fellow. Back

Received February 7, 2000.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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