Regulation of prohormone convertase 1 (PC1) by thyroid hormone

Qiao-Ling Li1,2, Erik Jansen4, Gregory A. Brent3, and Theodore C. Friedman1,2

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha 1 (TRalpha 1) and retinoid X receptor-beta (RXRbeta ) proteins and GH3 nuclear extracts demonstrated that the region from -10 to +19 bp of the hPC1 promoter bound TRalpha 1 as both a monomer and a homodimer and bound TRalpha 1/RXRbeta 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 alpha -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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 TRalpha 1, TRalpha 2, and TRbeta 1 were cloned into a pCDM8 expression vector (Invitrogen) and were used for cotransfection in JEG-3 and CV-1 cells.

Mutational analysis of the proximal promoter elements was done using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) according to the manufacturer's protocol. The -82- to +19-bp hPC1 promoter-luciferase construct was used as a template to generate a mutated construct. The oligonucleotide used was 5'-agggctggggctAAactcagccATTagaccgaagcg (upper-case letters indicate mutated nucleotides). Site-specific mutations were verified by sequencing.

GH3 cells were plated in growth medium in 6-well plates and allowed to adhere overnight. Cells were then placed in OPTI-MEM (GIBCO BRL) medium and overlaid with a mixture of DNA/cationic liposomes (lipofectamine, GIBCO BRL). Cells were incubated with DNA (3 µg/well) for 6 h, and the medium was changed to DMEM serum-free medium for overnight incubation, followed by treatment of cells with either T3 (10-8 M), 9-cis-retinoic acid (9-cis-RA, 10-7 M), or their combination for 24 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 (Wallace, Gaithersburg, MD) in the presence of 0.8 mM ATP and 0.3 mM D-luciferin. Integrated light emission over 15 s was measured. All transfections were done in triplicate and were repeated at least three times.

To study the effects of TR isoforms on the T3 regulation of PC1, 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 hPC1 promoter-luciferase constructs, 0.5 µg of TR, and 0.5 µg of carrier plasmid were used for transfection. RSV0-beta -galactosidase (beta -gal) plasmid (1 µg) was included in the transfection to measure transfection efficiency (32). Six hours after transfection, the cells were incubated in the presence or absence of T3 (10-8 M) for 24 h. Luciferase activity was divided by beta -gal activity to correct for transfection efficiency. All experiments were performed in duplicate plates, and each experiment was repeated three to five times.

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 [gamma -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 TRalpha 1 (0.01-0.1 µg, Santa Cruz Biotechnology, Santa Cruz, CA), or RXRbeta (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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 1.   Plasma thyroid-stimulating hormone (TSH) levels and Northern blot analysis of pituitary prohormone convertase (PC)1 mRNA in rats receiving 6-n-propyl-2-thiouracil (PTU) and triiodo-L-thyronine (T3). Plasma TSH levels from control (CON), PTU-treated (PTU) and PTU plus T3-treated rats (PTU/T3; A) representative Northern blot analysis of PC1 mRNA in anterior pituitary (B) of rats receiving PTU and T3 treatment. Hybridization with cyclophilin (Cyclo) was used as an internal control to correct for differences in RNA loading. The quantitative result of the PC1 signal after correction for cyclophilin) is expressed as means ± SE induction (n = 4) over the control. *P < 0.05 vs. control; **P < 0.005 vs. group with PTU treatment; ***P < 0.0001 vs. control.

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.


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of T3 and 9-cis-retinoic acid (9-cis-RA) on human PC1 (hPC1) promoter activity. GH3 cells were transiently transfected with hPC1-luciferase (-701 to +216 bp), rat GH (rGH)-luciferase, human thyrotropin-releasing hormone (hTRH)-luciferase and thymidine kinase (TK)-luciferase constructs using DNA cationic/liposomes method. After transfection and serum starvation, the cells were treated with either T3 (10-8 M), 9-cis-RA (10-7 M), or their combinations for 24 h. Cells were then harvested for luciferase assay as described. Results are expressed as means ± SE of luciferase activity from 3 independent experiments. *P < 0.05 vs. control.

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.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 3.   Deletion analysis to localize the putative thyroid hormone response elements in hPC1 promoter. Different hPC1 promoter constructs were transfected into GH3 cells and incubated in the presence or absence of T3 (10-8 M) for 24 h and then harvested for luciferase (Luc) assay. Results are expressed as means ± SE of luciferase activity with and without T3 from a representative experiment. The experiment was repeated on 3 independent occasions. *P < 0.05 vs. control for the 3 independent experiments.

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 TRalpha 1 or TRbeta 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 TRalpha 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 TRalpha 1 or TRbeta 1 isoform preference in the regulation of hPC1 promoter in the transient transfection system used in our study.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 4.   Thyroid hormone receptor (TR) isoforms on the repressive effect of T3 on hPC1 promoter in CV-1 and JEG-3 cells. CV-1 and JEG-3 cells were transfected with hPC1 -701 to +216-bp promoter construct with different TR isoforms for 24 h. Cells were then incubated in the presence or absence of T3 (10-8 M) for another 24 h before they were harvested for luciferase assay. Results are expressed as means ± SE of luciferase activity vs. control (without T3). *P < 0.05 vs. control.

Binding of purified TRalpha 1 and RXRbeta 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 TRalpha 1 and RXRbeta 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 TRalpha 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 TRalpha 1 (lane 2) did not show any binding to either DR4 or hPC1 fragments. RXR was then added to a low concentration of TRalpha 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.


View larger version (37K):
[in this window]
[in a new window]
 
Fig. 5.   Binding of hPC1 -82 to +19-bp fragment to purified TRalpha 1 and retinoid X receptor-beta (RXRbeta ). Electrophoretic mobility shift assays (EMSAs) were performed using the double-stranded direct-repeat 4 (DR4) element (A) corresponding to direct repeat of consensus half-site AGGTCA separated by 4 nucleotides and the hPC1 -82- to +19-bp fragment synthesized by PCR (B). The monomer (M), homodimer (HOD), heterodimer (HED), and multimer (MULT) bindings are indicated by arrows. Lane 1, probe alone; lane 2, probe and 0.1 µg of TRalpha 1; lane 3, probe, TRalpha 1, and 50-fold molar excess of cold DR4 (in A) or hPC1 fragment (in B); lane 4, probe, TRalpha 1, and 200-fold molar excess of nonspecific competitor (NON); lane 5, probe and 0.01 µg of TRalpha 1; lane 6, probe, TRalpha 1, and RXRbeta ; lane 7, probe, TRalpha 1, RXRbeta , and anti-TRalpha 1 antibody (Ab); lane 8, probe, TRalpha 1, RXRbeta , and anti-RXRbeta antibody; lane 9, probe and RXRbeta alone.

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 TRalpha 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 TRalpha 1 protein were included (Fig. 6B).


View larger version (49K):
[in this window]
[in a new window]
 
Fig. 6.   Binding of two oligonucleotides selected from -10 to +19 bp (O#1) and -37 to -18 bp (O#2) of the hPC1 promoter to purified TRalpha 1 and the ability of those oligonucleotides to compete with labeled DR4 for binding to GH3 nuclear extracts. A: sequences of two oligonucleotides (O#1 and O#2) selected from hPC1 promoter. B: probes were incubated with increasing amounts of TRalpha 1 protein (0, 0.01, 0.04, 1.0 µg). C: DR4 probe was incubated with GH3 nuclear extract, and binding of GH3 nuclear protein to DR4 was competed with increasing amounts of O#1 and O#2. Lane 1, probe alone; lane 2, probe and GH3 nuclear extract (NE); lane 3, probe, GH3 nuclear extract, and 200-fold molar excess of cold nonspecific competitor (NON); lane 4, probe, GH3 nuclear extract, and 5-fold cold DR4; lane 5, probe, GH3 nuclear extract, and anti-TRalpha 1 antibody; lanes 6-11, 200-, 20- and 5-fold molar excess of cold O#1 and O#2.

It has been demonstrated that specific nuclear proteins can enhance the TR binding to certain DNA elements (24). To confirm that the binding of O#1 from hPC1 promoter observed above with purified TRalpha 1 protein reflects the binding within GH3 cells, we performed EMSAs with double-stranded, unlabeled O#1 and O#2 to compete for the binding of GH3 cell nuclear extracts to the DR4 probe. As shown in Fig. 6C, a single binding band (indicated by a solid arrow) was observed when the DR4 probe was incubated with GH3 nuclear extracts (lane 2). Five molar excess of unlabeled DR4 (lane 4), but not 200 molar excess of nonspecific competitor (lane 3), completely abolished the binding. TRalpha 1 antibody diminished the intensity of the band (lane 5). These results suggest the specificity of TR proteins in GH3 nuclear extract binding to DR4 element. When excess of the two hPC1 oligonucleotides (200-, 20-, 5-fold excess) competing for DR4 binding to GH3 nuclear extracts was examined, the addition of O#1, but not O#2, efficiently inhibited the binding, the 200-fold excess of O#1 completely abolishing the binding (lane 6). Taken together, these results suggest that the binding of O#1 to purified TRalpha 1 is likely to reflect the binding occurring within GH3 cells, and the region between -10 and +19 bp on hPC1 promoter is likely responsible for the T3 negative regulation of the hPC1 promoter.

EMSAs using wild-type and mutant oligonucleotides with TRalpha 1 and RXRbeta proteins. To further investigate whether TR/RXR interaction occurred in O#1, EMSAs were carried out using purified TRalpha 1 and RXRbeta proteins with wild-type (WT) and mutant (Mut) oligonucleotides as probes (Fig. 7A). As indicated in Fig. 7B, when higher amounts of TRalpha 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 TRalpha 1 antibody supershifted the binding (lane 4), which suggests the specificity of the TRalpha 1 and O#1 interaction. In the lane (lane 5) with a low (0.01 µg) amount of TRalpha 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 TRalpha 1 and RXR were added to the reactions, heterodimer binding, but not multimer binding, was observed (Fig. 7C, lane 7).


View larger version (43K):
[in this window]
[in a new window]
 
Fig. 7.   Binding of wild-type (WT) and mutant (Mut) oligonucleotides to TRalpha 1 and RXRbeta proteins. A: sequences of WT and Mut O#1. The mutated nucleotides are indicated by underlining. B: the WT PC1 oligonucleotide was used as a probe for EMSAs; exposure time is longer than that of the EMSA in Fig. 6B. C: the Mut oligonucleotide was used as a probe for EMSAs; M, HOD, HED, and MULT bindings are indicated by arrows. Lane 1, probe alone; lane 2, probe and 0.1 µg of TRalpha 1; lane 3, probe, TRalpha 1, and T3 (10-6 M); lane 4, probe, TRalpha 1, and anti-TRalpha 1 antibody; lane 5, probe and 0.01 µg of TRalpha 1; lane 6, probe and RXRbeta alone; lane 7, probe, TRalpha 1, and RXRbeta ; lane 8, probe, TRalpha 1, RXRbeta , and T3 (10-6 M); lane 9, probe, TRalpha 1, RXRbeta , and anti-TRalpha 1 antibody. D: luciferase activity in the presence and absence of T3 for WT and Mut -82- to +19-bp hPC1 promoter construct. Reduced but still significant inhibition by T3 was obtained with the Mut construct. *P < 0.05 vs. without T3.

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).


    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 (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 RXRbeta ), 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 TRalpha 1 and TRbeta 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 TRalpha 1 and TRbeta 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 TRalpha 1 or TRbeta 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), TSHbeta subunit and glycoprotein-alpha subunit (5), exhibit variable half-site sequences, which may or may not contain consensus TRE sequences. EMSAs using purified TRalpha 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 TRalpha 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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aronin, N, Coslovsky R, and Chase K. Hypothyroidism increases substance P concentrations in the heterotopic anterior pituitary. Endocrinology 122: 2911-2914, 1988[Abstract].

2.   Birch, NP, Tracer HL, Hakes DJ, and Loh YP. Coordinate regulation of mRNA levels of pro-opiomelanocortin and the candidate processing enzymes PC2 and PC3, but not furin, in the rat pituitary intermediate lobe. Biochem Biophys Res Commun 179: 1311-1319, 1991[ISI][Medline].

3.   Bloomquist, BT, Eipper BA, and Mains RE. Prohormone-converting enzymes: regulation and evaluation of function using antisense RNA. Mol Endocrinol 5: 2014-2024, 1991[Abstract].

4.   Brent, GA, Larsen PR, Harney JW, Koenig RJ, and Moore DD. Functional characterization of the rat growth hormone promoter elements required for induction by thyroid hormone with and without a co-transfected beta type thyroid hormone receptor. J Biol Chem 264: 178-182, 1989[Abstract/Free Full Text].

5.   Burnside, J, Darling DS, Carr FE, and Chin WW. Thyroid hormone regulation of the rat glycoprotein hormone alpha-subunit gene promoter activity. J Biol Chem 264: 6886-6891, 1989[Abstract/Free Full Text].

6.   Day, R, Schafer MK-H, Watson SJ, Chretien M, and Seidah NG. Distribution and regulation of the prohormone convertases PC1 and PC2 in the rat pituitary. Mol Endocrinol 6: 485-497, 1992[Abstract].

7.   Dignam, JD. Preparation of extracts from higher eukaryotes. Methods Enzymol 182: 194-203, 1990[ISI][Medline].

8.   Feng, P, Li QL, Satoh T, and Wilber JF. Ligand (T3) dependent and independent effects of thyroid hormone receptors upon human TRH gene transcription in neuroblastoma cells. Biochem Biophys Res Commun 200: 171-177, 1994[ISI][Medline].

9.   Force, WR, Tillman JB, Sprung CN, and Spindler SR. Homodimer and heterodimer DNA binding and transcriptional responsiveness to triiodothyronine (T3) and 9-cis-retinoic acid are determined by the number and order of high affinity half-sites in a T3 response element. J Biol Chem 269: 8863-8871, 1994[Abstract/Free Full Text].

10.   Furuta, M, Yano H, Zhou A, Rouille Y, Holst JJ, Carroll R, Ravazzola M, Orci L, Furuta H, and Steiner DF. Defective prohormone processing and altered pancreatic islet morphology in mice lacking active SPC2. Proc Natl Acad Sci USA 94: 6646-6651, 1997[Abstract/Free Full Text].

11.   Hakes, DJ, Birch NP, Mezey E, and Dixon JE. Isolation of two complementary deoxyribonucleic acid clones from a rat insulinoma cell line based on similarities to Kex2 and furin sequences and the specific localization of each transcript to endocrine and neuroendocrine tissues in rats. Endocrinology 129: 3053-3063, 1991[Abstract].

12.   Harbers, M, Wahlstrom GM, and Vennstrom B. Transactivation by the thyroid hormone receptor is dependent on the spacer sequence in hormone response elements containing directly repeated half-sites. Nucleic Acids Res 24: 2252-2259, 1996[Abstract/Free Full Text].

13.   Herman-Bonert, V, Friedman TC, and Braunstein GD. The thyroid gland. In: Cecil Essentials of Medicine, edited by Andreoli TE. Philadelphia, PA: W. B. Saunders, 1997, p. 487-496.

14.   Hodin, RA, Lazar MA, Wintman BI, Darling DS, Koenig RJ, Larsen PR, Moore DD, and Chin WW. Identification of a thyroid hormone receptor that is pituitary-specific. Science 244: 76-79, 1989[ISI][Medline].

15.   Hooi, SC, Koenig JI, Abraczinskas DR, and Kaplan LM. Regulation of anterior pituitary galanin gene expression by thyroid hormone. Brain Res Mol Brain Res 51: 15-22, 1997[ISI][Medline].

16.   Jackson, RS, Creemers JW, Ohagi S, Raffin-Sanson ML, Sanders L, Montague CT, Hutton JC, and O'Rahilly S. Obesity and impaired prohormone processing associated with mutations in the human prohormone convertase 1 gene. Nat Genet 16: 303-306, 1997[ISI][Medline].

17.   Jansen, E, Ayoubi TA, Meulemans SM, and Van de Ven WJ. Neuroendocrine-specific expression of the human prohormone convertase 1 gene. Hormonal regulation of transcription through distinct cAMP response elements. J Biol Chem 270: 15391-15397, 1995[Abstract/Free Full Text].

18.   Jansen, E, Ayoubi TA, Meulemans SM, and Van de Ven WJ. Cell type-specific protein-DNA interactions at the cAMP response elements of the prohormone convertase 1 promoter. Evidence for additional transactivators distinct from CREB/ATF family members. J Biol Chem 272: 2500-2508, 1997[Abstract/Free Full Text].

19.   Jansen, E, Ayoubi TA, Meulemans SM, and Van De Ven WJ. Regulation of human prohormone convertase 2 promoter activity by the transcription factor EGR-1. Biochem J 328: 69-74, 1997[ISI][Medline].

20.   Johanning, K, Juliano MA, Juliano L, Lazure C, Lamango NS, Steiner DF, and Lindberg I. Specificity of prohormone convertase 2 on proenkephalin and proenkephalin-related substrates. J Biol Chem 273: 22672-22680, 1998[Abstract/Free Full Text].

21.   Johnson, RC, Darlington DN, Hand TA, Bloomquist BT, and Mains RE. PACE4: a subtilisin-like endoprotease prevalent in the anterior pituitary and regulated by thyroid status. Endocrinology 135: 1178-1185, 1994[Abstract].

22.   Jones, PM, Ghatei MA, Steel J, O'Halloran D, Gon G, Legon S, Burrin JM, Leonhardt U, Polak JM, and Bloom SR. Evidence for neuropeptide Y synthesis in the rat anterior pituitary and the influence of thyroid hormone status: comparison with vasoactive intestinal peptide, substance P, and neurotensin. Endocrinology 125: 334-341, 1989[Abstract].

23.   Kakizawa, T, Miyamoto T, Kaneko A, Yajima H, Ichikawa K, and Hashizume K. Ligand-dependent heterodimerization of thyroid hormone receptor and retinoid X receptor. J Biol Chem 272: 23799-23804, 1997[Abstract/Free Full Text].

24.   Koenig, RJ. Thyroid hormone receptor coactivators and corepressors. Thyroid 8: 703-713, 1998[ISI][Medline].

25.   Lam, KS, Lechan RM, Minamitani N, Segerson TP, and Reichlin S. Vasoactive intestinal peptide in the anterior pituitary is increased in hypothyroidism. Endocrinology 124: 1077-1084, 1989[Abstract].

26.   Langlois, MF, Zanger K, Monden T, Safer JD, Hollenberg AN, and Wondisford FE. A unique role of the beta-2 thyroid hormone receptor isoform in negative regulation by thyroid hormone. Mapping of a novel amino-terminal domain important for ligand-independent activation. J Biol Chem 272: 24927-24933, 1997[Abstract/Free Full Text].

27.   Lazar, MA. Thyroid hormone receptors: multiple forms, multiple possibilities. Endocr Rev 14: 184-193, 1993[ISI][Medline].

28.   Lee, HW, Klein LE, Raser J, and Eghbali-Webb M. An activator protein-1 (AP-1) response element on pro alpha1(l) collagen gene is necessary for thyroid hormone-induced inhibition of promoter activity in cardiac fibroblasts. J Mol Cell Cardiol 30: 2495-2506, 1998[ISI][Medline].

29.   Lezoualc'h, F, Hassan AH, Giraud P, Loeffler JP, Lee SL, and Demeneix BA. Assignment of the beta-thyroid hormone receptor to 3,5,3'-triiodothyronine-dependent inhibition of transcription from the thyrotropin-releasing hormone promoter in chick hypothalamic neurons. Mol Endocrinol 6: 1797-1804, 1992[Abstract].

30.   Li, Q-L, Jansen E, Brent GA, Naqvi S, Wilber JF, and Friedman TC. Interactions between the prohormone convertase 2 (PC2) promoter and the thyroid hormone receptor. Endocrinology 141: 3256-3266, 2000[Abstract/Free Full Text].

31.   Li, Q-L, Feng P, Koch C, Shi ZX, and Wilber JF. Reversal of TR-T3 inhibition of the hTRH gene by excess TR ligand-binding domain: evidence for novel accessory protein. Thyroid 6: 233-236, 1996[ISI][Medline].

32.   Li, Q-L, Feng P, Satoh T, Shi ZX, Wang R, Weintraub BD, and Wilber JF. Regulation of the human TRH (hTRH) gene by human thyroid hormone receptor beta 1 (hTR beta 1) mutants. Endocr Res 23: 297-309, 1997[ISI][Medline].

33.   Li, Q-L, Jansen E, and Friedman TC. Regulation of prohormone convertase 1 (PC1) by gp130-related cytokines. Mol Cell Endocrinol 158: 143-152, 1999[ISI][Medline].

34.   Li, Q-L, Yano H, Ren SG, Li X, Friedman TC, and Melmed S. Leukemia inhibitory factor (LIF) modulates pro-opiomelanocortin (POMC) gene regulation in stably-transfected AtT-20 cells over-expressing LIF. Endocr J 7: 325-330, 1998.

35.   Misiti, S, Schomburg L, Yen PM, and Chin WW. Expression and hormonal regulation of coactivator and corepressor genes. Endocrinology 139: 2493-2500, 1998[Abstract/Free Full Text].

36.   Ohagi, S, LaMendola J, LeBeau MM, Espinosa R, III, Takeda J, Smeekens SP, Chan SJ, and Steiner DF. Identification and analysis of the gene encoding human PC2, a prohormone convertase expressed in neuroendocrine tissues. Proc Natl Acad Sci USA 89: 4977-4981, 1992[Abstract].

37.   Oppenheimer, JH, Schwartz HL, and Strait KA. The molecular basis of thyroid hormone actions. In: Werner and Ingbar's The Thyroid, edited by Braverman LE, and Utiger RD. Philadelphia, PA: Lippincott-Raven, 1996, p. 162-184.

38.   O'Rahilly, S, Gray H, Humphreys PJ, Krook A, Polonsky KS, White A, Gibson S, Taylor K, and Carr C. Brief report: impaired processing of prohormones associated with abnormalities of glucose homeostasis and adrenal function. N Engl J Med 333: 1386-1390, 1995[Free Full Text].

39.   Ouafik, L, May V, Saffen DW, and Eipper BA. Thyroid hormone regulation of peptidylglycine alpha-amidating monooxygenase expression in anterior pituitary gland. Mol Endocrinol 4: 1497-1505, 1990[Abstract].

40.   Satoh, T, Yamada M, Iwasaki T, and Mori M. Negative regulation of the gene for the preprothyrotropin-releasing hormone from the mouse by thyroid hormone requires additional factors in conjunction with thyroid hormone receptors. J Biol Chem 271: 27919-27926, 1996[Abstract/Free Full Text].

41.   Scott, RE, Wu-Peng XS, Yen PM, Chin WW, and Pfaff DW. Interactions of estrogen- and thyroid hormone receptors on a progesterone receptor estrogen response element (ERE) sequence: a comparison with the vitellogenin A2 consensus ERE. Mol Endocrinol 11: 1581-1592, 1997[Abstract/Free Full Text].

42.   Seidah, NG, Benjannet S, Hamelin J, Mamarbachi AM, Basak A, Marcinkiewicz J, Mbikay M, Chretien M, and Marcinkiewicz M. The subtilisin/kexin family of precursor convertases. Emphasis on PC1, PC2/7B2, POMC and the novel enzyme SKI-1. Ann NY Acad Sci 885: 57-74, 1999[Abstract/Free Full Text].

43.   Seidah, NG, and Chretien M. Proprotein and prohormone convertases: a family of subtilases generating diverse bioactive polypeptides. Brain Res 848: 45-62, 1999[ISI][Medline].

44.   Seidah, NG, Marcinkiewicz M, Benjannet S, Gaspar L, Beaubien G, Mattei MG, Lazure C, Mbikay M, and Chretien M. Cloning and primary sequence of a mouse candidate prohormone convertase PC1 homologous to PC2, furin, and Kex2: distinct chromosomal localization and messenger RNA distribution in brain and pituitary compared to PC2. Mol Endocrinol 5: 111-122, 1991[Abstract].

45.   Snyder, PJ. The pituitary in hypothyroidism. In: Werner and Ingbar's The Thyroid, edited by Braverman LE, and Utiger RD. Philadelphia, PA: Lippincott-Raven, 1996, p. 836-840.

46.   Snyder, PJ. The pituitary in thyrotoxicosis. In: Werner and Ingbar's The Thyroid, edited by Braverman LE, and Utiger RD. Philadelphia, PA: Lippincott-Raven, 1996, p. 653-655.

47.   Tagami, T, Gu WX, Peairs PT, West BL, and Jameson JL. A novel natural mutation in the thyroid hormone receptor defines a dual functional domain that exchanges nuclear receptor corepressors and coactivators. Mol Endocrinol 12: 1888-1902, 1998[Abstract/Free Full Text].

48.   Takeshita, A, Yen PM, Ikeda M, Cardona GR, Liu Y, Koibuchi N, Norwitz ER, and Chin WW. Thyroid hormone response elements differentially modulate the interactions of thyroid hormone receptors with two receptor binding domains in the steroid receptor coactivator-1. J Biol Chem 273: 21554-21562, 1998[Abstract/Free Full Text].

49.   Takumi, I, Steiner DF, Sanno N, Teramoto A, and Osamura RY. Localization of prohormone convertases 1/3 and 2 in the human pituitary gland and pituitary adenomas: analysis by immunohistochemistry, immunoelectron microscopy, and laser scanning microscopy. Mod Pathol 11: 232-238, 1998[ISI][Medline].

50.   Toyoda, N, Zavacki AM, Maia AL, Harney JW, and Larsen PR. A novel retinoid X receptor-independent thyroid hormone response element is present in the human type 1 deiodinase gene. Mol Cell Biol 15: 5100-5112, 1995[Abstract].

51.   Udupi, V, Townsend C, Jr, and Greeley G, Jr. Stimulation of prohormone convertase-1 mRNA expression by second messenger signaling systems. Biochem Biophys Res Commun 246: 463-465, 1998[ISI][Medline].

52.   Xu, J, Thompson KL, Shephard LB, Hudson LG, and Gill GN. T3 receptor suppression of Sp1-dependent transcription from the epidermal growth factor receptor promoter via overlapping DNA-binding sites. J Biol Chem 268: 16065-16073, 1993[Abstract/Free Full Text].

53.   Yen, PM, Brubaker JH, Apriletti JW, Baxter JD, and Chin WW. Roles of 3,5,3'-triiodothyronine and deoxyribonucleic acid binding on thyroid hormone receptor complex formation. Endocrinology 134: 1075-1081, 1994[Abstract].

54.   Yen, PM, Ikeda M, Wilcox EC, Brubaker JH, Spanjaard RA, Sugawara A, and Chin WW. Half-site arrangement of hybrid glucocorticoid and thyroid hormone response elements specifies thyroid hormone receptor complex binding to DNA and transcriptional activity. J Biol Chem 269: 12704-12709, 1994[Abstract/Free Full Text].

55.   Zhou, A, Webb G, Zhu X, and Steiner DF. Proteolytic processing in the secretory pathway. J Biol Chem 274: 20745-20748, 1999[Free Full Text].


Am J Physiol Endocrinol Metab 280(1):E160-E170
0193-1849/01 $5.00 Copyright © 2001 the American Physiological Society