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Endocrinology, doi:10.1210/en.2005-1609
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Endocrinology Vol. 147, No. 6 2705-2716
Copyright © 2006 by The Endocrine Society

Thyroid Hormones Selectively Regulate the Posttranslational Processing of Prothyrotropin-Releasing Hormone in the Paraventricular Nucleus of the Hypothalamus

Mario Perello, Theodore Friedman, Veronica Paez-Espinosa, Xiaoxiong Shen, Ronald C. Stuart and Eduardo A. Nillni

Division of Endocrinology, Department of Medicine Brown University/Rhode Island Hospital (M.P., R.C.S., E.A.N.), Providence, Rhode Island 02903; Division of Endocrinology, Department of Medicine, Charles R. Drew University of Medicine and Sciences-University of California School of Medicine (T.F., V.P.-E., X.S.), Los Angeles, California 90059; and Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University (E.A.N.), Providence, Rhode Island 02903

Address all correspondence and requests for reprints to: Dr. Eduardo A. Nillni, Division of Endocrinology, Brown Medical School/Rhode Island Hospital, 55 Claverick Street, Fourth Floor, Room 430, Providence, Rhode Island 02903. E-mail: eduardo_nillni{at}brown.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Over the last few years, our laboratory has demonstrated that different physiological conditions or stressors affect the posttranslational processing of hypophysiotropic and nonhypophysiotropic proTRH and, consequently, the output of TRH and other proTRH-derived peptides. These alterations in proTRH processing are generally associated with parallel changes in the levels of two members of the family of prohormone convertases 1/3 and 2 (PC1/3 and PC2). An important regulator of proTRH is thyroid hormone, which is the peripheral end product of the hypothalamic (TRH)-pituitary (TSH)-thyroid (T3/4) (HPT) axis. In this study we investigated the effect of thyroid status on the processing of proTRH inside and outside the HPT axis. Our data showed that high levels of thyroid hormone down-regulated PC1/3 and PC2 and TRH synthesis, which led to an accumulation of intermediate forms of proTRH processing. Conversely, low levels of thyroid hormone up-regulated proTRH synthesis and PC1/3 and PC2 levels. Control of the activity of PCs and proTRH processing occurred specifically in the paraventricular nucleus, whereas no change due to thyroid status was found in the lateral hypothalamus or preoptic area. The posttranslational regulation of proTRH processing in the paraventricular nucleus by thyroid status is a novel aspect of the regulation of the HPT axis, which may have important implications for the pathophysiology of hypo- and hyperthyroidism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE HYPOTHALAMIC-PITUITARY-THYROID (HPT) axis plays an important role in the maintenance of metabolic homeostasis in response to alterations in the external environment. The hypothalamic releasing factor, TRH, is recognized as a key hormone responsible for HPT regulation. TRH is synthesized from a larger inactive precursor, proTRH (26 kDa), through posttranslationally modifications in the paraventricular nucleus (PVN) (primarily in the parvocellular division) of the hypothalamus by the action primarily of the prohormone convertase 1/3 (PC1/3) and secondarily by PC2 (1, 2, 3) (Fig. 1Go). The intermediate products of proTRH generated from these enzymatic cleavages are subjected to additional modifications by exopeptidases, such as carboxypeptidase E or carboxypeptidase D, to remove the C-terminal basic amino acids (4). TRH-Gly, the immediate precursor to TRH (pGlu-His-Pro-NH2; thyroliberin), is then amidated at their carboxyl terminus by the action of peptidylglycine {alpha}-amidating monooxygenase enzyme (5).


Figure 1
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  		FIG. 1. Diagrammatic representation of rat proTRH and its processing to TRH and non-TRH peptides (10 ). It also indicates the sites where PC1 and PC2 produce their enzymatic cleavages. Below the full length of the proTRH precursor are depicted the N-terminal peptides recognized by the anti-preproTRH83–106 (pEH24) antibody, and above the full length of the proTRH precursor is depicted the only peptide recognized by the anti-preproTRH238–255 (pYE17) antibody.

 
Hypophysiotropic TRH neurons in the PVN project to the median eminence (ME), where they are in close proximity to the capillaries of the hypophysial-portal system. TRH released in these capillaries stimulates biosynthesis and secretion of TSH from the pituitary (6, 7), which, in turn, stimulates biosynthesis of the thyroid hormones, T4 and T3, and their release from the thyroid gland. The maintenance of euthyroidism is dependent on a highly regulated balance of positive and negative feedback, in which TRH positively regulates TSH (6, 7), and thyroid hormones suppress preproTRH expression and TSH secretion (8). ProTRH is also abundantly expressed in hypothalamic neurons outside the PVN and in extrahypothalamic brain regions. This extrahypophysiotropic TRH is believed to function as a neuromodulator of known neurotransmitters (8, 9). It probably has an important role in appetite control, arousal and sleep, cognition, locomotion antinociception, thermoregulation, and psychological function (10). Little is known, however, about the regulation of extrahypophysiotropic TRH and its biosynthesis by thyroid hormone status.

The important role of PC1/3 and PC2 in hormonal biosynthesis has been elucidated by the studies of PC1/3- and PC2-null mice and in a patient with defective PC1/3. PC1/3-null mice are small due to impaired proGHRH processing and are hyperglycemic due to impaired proinsulin processing (11, 12). ProTRH processing in PC1/3-null mice showed a substantial decrease in the biosynthesis of all proTRH-derived peptides, including TRH and its proform, TRH-Gly, whereas PC2-null mice showed a minor defect in proTRH processing (Nillni, E. A., and D. Steiner, unpublished observations). PC2-null mice have absent proglucagon processing, leading to hypoglycemia, and also have impaired proinsulin processing (13). Proopiomelanocortin, proenkephalin, and prodynorphin processing are blunted (14, 15). A patient lacking PC1/3 had severe childhood-onset obesity, postprandial hypoglycemia, infertility, and low levels of ACTH and cortisol with elevated levels of proopiomelanocortin (16, 17). The finding that knockout mice have processing profiles that are expected from the known biochemistry of PC1/3 and PC2 supports the belief that these two enzymes are necessary and sufficient for the biosynthesis of most hormones/neuropeptides.

We have previously localized regions on PC1/3 and PC2 human promoters that contain putative negative thyroid response elements and have also shown that T3 negatively regulates PC1/3 and/or PC2 expression in rat GH3 cells, rat anterior pituitary, hypothalamus, and cerebral cortex (18, 19, 20, 21). These antecedents open the possibility that thyroid hormones could regulate PC1/3 and PC2 expression in specific hypothalamic nuclei directly regulating proTRH processing. Therefore, we altered the thyroid hormone status in rats and examined the effects on proTRH processing in the PVN and other selected extrahypophysiotropic sites. We also examined the effects of thyroid hormone status on PC1/3 and PC2 enzyme levels. Our results support the hypothesis that high levels of thyroid hormone down-regulate TRH biosynthesis, which is coupled with the down-regulation of PC1/3 and PC2 in the PVN. Conversely, low levels of thyroid hormone up-regulate TRH biosynthesis and PC1/3 levels. Such regulation may have important implications in the pathophysiology of hypo- and hyperthyroidism.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and treatments
Adult male Sprague Dawley rats (250–270 g) were housed in a room with controlled light, temperature, and humidity. The experimental protocols and euthanasia procedures were reviewed and approved by the institutional animal care and use committees of Rhode Island Hospital/Brown University and Charles R. Drew University of Medicine and Sciences-University of California-Los Angeles School of Medicine. Three groups of 12 animals each were treated as follows. In the control group (CTR), implantation of a placebo pellet with normal chow; in the hyperthyroid group (T4), implantation of a thyroid hormone (L-T4) pellet (15 mg; Innovative Research of America, Sarasota, FL) with normal chow; in the hypothyroid group [propylthiouracil (PTU)], and implantation of a placebo pellet with chow containing low iodine and 0.15% PTU (Harlan Teklad, Madison, WI). Pellet implantations were performed under the skin on the lateral side of the neck of the animal. PTU blocks thyroidal hormone synthesis by inhibition of thyroid peroxidase activity and inhibits type I iodothyronine deiodinase in liver and kidney, important sites for the peripheral production of T3 (22). Animals had free access to food and water and were killed by decapitation 21 d after treatment.

Samples
Blood was collected immediately after decapitation for TSH, T3 and T4 analysis. The PVN, ME, lateral hypothalamus (Lh) and the preoptic area (POA) regions were rapidly removed after decapitation by surgical dissection and subjected to acid peptide extraction with 2 N acetic acid freshly supplemented with a protease inhibitor mixture [4-(2-aminoethyl)-benzenesulfonylfluoride.HCl (AEBSF), pepstatin A, E64, bestatin, leupeptin, and aprotinin; Sigma-Aldrich Corp., St Louis, MO] as previously described (23). Cell extracts were then heated at 95 C for 10 min and sonicated, and cell disruption was performed by 15 strokes using a Dounce homogenizer (Fisher Scientific, Pittsburgh, PA). After cell disruption, the samples were centrifuged at 15,000 rpm for 30 min, and the supernatant was subjected to specific RIA or further purified by fractionation onto SDS-PAGE, followed by gel slicing, elution, and RIA.

RIA analyses
All RIAs and antibodies used in this study were developed in our laboratory and fully described previously (1). The tracers were iodinated using the chloramine-T oxidation-reduction method, followed by HPLC purification. The RIA used to measure TRH was performed as a standard in our laboratory using a specific TRH antiserum that recognizes only mature TRH peptide. For the detection of proTRH-derived peptides, we used anti-preproTRH83–106 (pEH24) and anti-preproTRH238–255 (pYE17) antiserum (23). The C-terminal antibody, anti-pYE17, only recognizes the end product, preproTRH208–255 (5.4 kDa), and the N-terminal antibody, anti-pEH24, recognizes preproTRH77–112 (4.8 kDa), preproTRH77–106 (3.8 kDa), and preproTRH83–106 (2.8 kDa; see Fig. 1Go). All RIAs were performed using the same volume of material in duplicate. The sensitivities of the TRH, pEH24, and pYE17 assays were 2.0, 40.0, and 15.0 pg/tube, respectively. The intra- and interassay variabilities were 5–6% and 9–12%, respectively.

TSH levels in rat serum were determined using a highly sensitive, double-antibody method developed by A. F. Parlow, National Hormone and Pituitary Program (Harbor-University of California-Los Angeles Medical Center, Torrance, CA) and previously used in our laboratory (24). The assay used highly purified rat TSH as the iodinated ligand, a guinea pig antirat TSH at a final tube dilution of 1:500,000 as the primary antibody, and a partially purified extract of rat pituitary containing TSH as the reference preparation. The cross-reactivity of either highly purified rat FSH or rat LH in this mouse TSH RIA was less than 1%. Displacement curves obtained by testing sera of hypothyroid rats in graded dilutions did not depart significantly from parallelism with displacement curves for the reference preparation. The sensitivity of the assay is such that TSH levels in hyperthyroid and euthyroid animals are completely distinguishable. The recovery of exogenous rat TSH activity added to rat serum was 80–100%. Serum T3 and T4 levels were determined according to the procedures and reagents provided by MP Biomedicals Diagnostic Division (Orangeburg, NY). The sensitivities of the T3 and T4 assays were 12.5 ng/dl and 1.2 µg/dl, and the intra- and interassay variabilities were approximately 5–7% and 10–11%, respectively. RIAs were performed without knowledge of the treatment status of the rat.

Sodium dodecyl sulfate-polyacrylamide gel fractionation of proTRH peptides
Eighty micrograms of total protein extracted in acetic acid from different hypothalamic regions of different groups were used in this experiment. The supernatants were evaporated using an ultracold speed vacuum system and then dissolved in sample buffer [0.0625 M Tris (pH 6.8), 1% sodium dodecyl sulfate, 15% glycerol, and 0.05% bromophenol blue], boiled for 5 min, and loaded onto a discontinuous tricine-polyacrylamide gel electrophoresis system (1.5 mm thick). A stacking gel was made to 3% cross-linking (acrylamide/bis-solution), and the separating gel was made to 6% cross-linking (acrylamide/bis-solution). The following molecular mass markers were used: carbonic anhydrase, 29.0 kDa; trypsin inhibitor, 20.4 kDa; lysozyme, 14.4 kDa; aprotinin 6.5 kDa; and insulin 2.87 (Bio-Rad Laboratories, Richmond, CA). After electrophoresis, gels were cut into 2-mm slices in a gel slicer (Hoeffer Scientific Instruments, San Francisco, CA). Peptides were extracted from the gel slices by incubation in 0.5 ml 2 N acetic acid for 18 h at 4 C. These fractions were evaporated using an ultracold speed vacuum system and then reconstituted in 0.5 ml of the buffer used in the pEH24 and pYE17 RIAs.

Western blot analysis
For Western blot analysis, each brain region (PVN, ME, POA and Lh) was dissected as described above, but the samples were collected and frozen separately for each animal. Microdissected samples were then homogenized with glass microhomogenizers (Wheaton, Milvale, NJ) on ice in extraction buffer [50 mM Tris-HCl (pH 7.2), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycolate, and 0.1% sodium dodecyl sulfate] and freshly supplemented with an inhibitor mixture. The final concentrations of inhibitor mixture in the extracted samples were as follows: phenylmethanylsulfonylfluoride, 100 µg/ml; leupeptin, 2 µg/ml; pepstatin, 100 µM; aprotinin, 2 µg/ml; and dithiothreitol, 2 mM. The samples were then centrifuged at 15,000 rpm for 30 min at 4 C, and the supernatant was removed and subjected to protein determination using the Bradford assay (Coomassie Protein Assay Reagent, Pierce Chemical Co., Rockford, IL). Twenty-five micrograms of total protein extracted in extraction buffer from different hypothalamic regions of different groups were used in this experiment. The supernatants were evaporated using an ultracold speed vacuum system, dissolved in sample buffer, and boiled for 5 min before SDS-PAGE. The samples were applied to 8% glycine-SDS-PAGE gels. The Precision Plus protein standards (dual color) were used as molecular weight markers (Bio-Rad Laboratories). After electrophoresis, proteins were electroblotted onto polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA) and blocked with 5% milk in Tris-buffered saline (50 mM Tris and 150 mM NaCl, pH 7.4). The membranes were probed overnight at 4 C with a 1:1000 dilution of anti-PC1/3 and anti-PC2 antiserum in 0.5% milk in Tris-buffered saline containing 0.1% Tween 20 as previously described (3). An alkaline phosphatase-linked goat antirabbit Ig secondary antibody (1:2000 final dilution) was used, and immunoreactive bands were visualized by an Immunostar assay as described by the manufacturer (Bio-Rad Laboratories).

Immunohistochemistry
For immunohistochemical experiments, three groups of animals were perfused and fixed under anesthesia with PBS, followed by 4% paraformaldehyde in PBS, and the brains were removed and postfixed in 4% paraformaldehyde overnight at 4 C. Sections (5 µm) were deparaffinized in xylene for 10 min and rehydrated by 3-min incubations in ethanol as follows: two incubations in 100% ethanol, followed by two incubations in 95% alcohol and one incubation in 75% alcohol. Once hydrated, sections were rinsed in tap water for 2 min and distilled water for 2 min. Endogenous peroxide activity was blocked by placing slides in 0.3% H2O2 for 30 min, followed by a 5-min rinse in PBS buffer. Slides were then processed for antigen detection using 10 mM sodium citrate buffer (pH 6.0). Slides were heated to boiling for 40 min, then cooled for 20 min and mounted on SuperFrost Plus (Fisher Scientific, Pittsburgh, PA). Localization of pro-TRH, PC1/3, and PC2 proteins was performed using the Vectastain Elite ABC kit (Vector Laboratories, Inc., Burlingame, CA) according to the manufacturer’s protocols. Primary polyclonal antibodies used for staining were directed against proTRH (pYE17, which recognizes both pro-TRH and C-terminal intermediate products; see Fig. 1Go) (10), PC1/3 or PC2 [both raised in rabbits against either a PC1-glutathione fusion protein or a PC2-glutathione fusion protein (25), provided by Dr. Nigel Birch (University of Auckland, Auckland, Australia) that recognizes proPC1/3 and active PC1/3 as well as proPC2 and active PC2; all diluted to 1:400]. Slides were incubated with primary antibodies for 60 min. Staining was visualized with the 3,3'-diaminobenzidine substrate kit (Vector Laboratories, Inc.). Quantification of the integrated intensity per area (integrated OD) of the positively staining neurons as well as the number of neurons per area staining with proTRH or PC1/3 and PC2 was performed using ImagePro Plus software (Media Cybernetics, Inc., Silver Spring, MD) under a light microscope. Two adjacent sections from each rat were averaged, and quantification for three rats for each group was performed.

Statistical analyses
The data are expressed as the mean ± SEM. Data for plasma hormone and peptide processing were analyzed by ANOVA, followed by Fisher’s test for comparison of different mean values. Significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Changes in thyroid status affect only hypophysiotropic TRH
As shown in Fig. 2AGo, serum T4 significantly decreased in PTU-treated rats (2.52 ± 0.10 µg/dl; n = 6) and increased in T4-treated rats (10.0 ± 0.83 µg/dl; P < 0.05; n = 6) compared with those in CTR animals (5.02 ± 0.17 µg/dl; P < 0.05; n = 6). Plasma T3 levels were also significantly increased by T4 treatment (125 ± 24 ng/dl; P < 0.05; n = 6) vs. CTR animals (75.8 ± 3.9 ng/dl; n = 6) and were undetectable in PTU-treated animals based on our assay sensitivity (<12.5 ng/dl). Consistent with these changes in thyroid hormones, we found increased plasma TSH levels in PTU-treated rats (18.9 ± 1.6 ng/ml; P < 0.01) vs. CTR rats (4.40 ± 0.9 ng/ml) and decreased plasma TSH levels in T4-treated rats (0.60 ± 0.10 ng/ml; P < 0.01) compared with CTR rats (Fig. 2BGo).


Figure 2
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  		FIG. 2. Effects of T4 and low iodine/PTU treatments on different parameters of the HPT axis. A and B, Comparative values by specific RIAs of serum T4 and TSH. C and D, Comparative values by specific RIAs of TRH in the PVN and ME. Values are the mean ± SEM. ANOVA was followed by multiple comparison using Fisher’s test. *, P < 0.05, significant difference at this level.

 
The concentration of TRH in the PVN was increased 38% in PTU-treated animals (1.21 ± 0.07 pg/µg protein; n = 6) compared with CTR animals (0.88 ± 0.09 pg/µg protein; P < 0.05; n = 6); T4-treated rats showed a decrease of 23% in TRH levels in the PVN (0.68 ± 0.03 pg/µg protein; P < 0.05; n = 6) vs. CTR rats (Fig. 2CGo). In addition, the concentration of immunoreactive TRH in the ME was decreased by 38% in PTU-treated animals (2.35 ± 0.41 pg/µg protein; n = 6) compared with CTR animals (3.80 ± 0.19 pg/µg protein; P < 0.05; n = 6); T4-treated rats showed an increase of 26% in TRH levels in ME (4.80 ± 0.26 pg/µg protein; P < 0.05 vs. CTR rats; n = 6; Fig. 2DGo).

Interestingly, TRH levels in the Lh were not significantly different among CTR animals (0.38 ± 0.06 pg/µg protein; n = 6), PTU-treated animals (0.29 ± 0.07 pg/µg protein; n = 6), and T4-treated rats (0.33 ± 0.06 pg/µg protein; n = 6). Similarly, TRH levels in the POA were not significantly different among CTR animals (0.87 ± 0.15 pg/µg protein; n = 6), PTU-treated animals (0.89 ± 0.17 pg/µg protein; n = 6), and T4-treated rats (0.79 ± 0.16 pg/µg protein; n = 6).

Hypothyroidism increased the biosynthesis and processing of proTRH in the PVN and their release from the ME
As depicted in Fig. 2DGo, the ME of hypothyroid animals had lower levels of TRH peptide compared with that of CTR animals, probably due to an increase in peptide release. In contrast, the PVN (Fig. 2CGo) of hypothyroid rats had higher TRH levels than that the CTR animals. To determine whether the increased levels of TRH in the PVN are due to an increase in the processing of the prohormone, we used two anti-proTRH-derived peptides antibodies. The first one, an antiserum made against the last 17 amino acids of the proTRH sequence (anti-pYE17), recognizes the 5.4-kDa end product peptide (proTRH208–255; Fig. 1Go) (23). The second antibody, made against one of the N-terminal end products of processing, pEH24 (preproTRH83–106), recognizes several intermediate forms, including approximately 4.8-kDa (propEH24), approximately 3.8-kDa (TRH-pEH24), and 2.75-kDa (pEH24) peptides (26). Figure 3Go, A and C, shows that the 5.4-kDa C-terminal peptide accumulated in the PVN, consistent with the accumulation of TRH (Fig. 2CGo), whereas the same peptide decreased in the ME similar to the decrease observed for the TRH peptide. We did not detect any changes in the 5.4-kDa peptide in iodine/PTU-treated animals vs. CTR in the POA and Lh (Fig. 3Go, B and D). Interestingly, we found a novel 2-kDa peptide in the Lh (Fig. 3BGo), probably a by-product of the 5.4-kDa peptide.


Figure 3
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  		FIG. 3. Low iodine/PTU treatment altered the biosynthesis and release of the C-terminal side of proTRH. This figure depicts an electrophoretic separation on a tricine-sodium dodecyl sulfate-polyacrylamide gel of PVN (A), Lh (B), ME (C), and POA (D) samples extracted from hypothyroid or CTR animals, followed by acid extraction of gel slices and RIA against pYE17 peptide. The molecular masses of the identified peaks are indicated based on the migration of standards and previously identified peaks using amino acid synthetic peptide standards (23 ). Each figure represents a typical profile of three independent experiments.

 
When we analyzed the processing of the N-terminal peptides using the anti-pEH24 antiserum, we found that the end products of processing accumulated in the PVN and decreased in the ME during iodine/PTU treatment (Fig. 4Go). We found that a small amount of the TRH-pHE24 form was present in the PVN, but was fully processed to pHE24 when it reached the ME (Fig. 4Go, A and C) (24). Analyses of the ratio between pro-pEH24 and pEH24 peptides revealed that the processing of N-terminal peptides was altered for PTU treatment vs. CTR in the PVN (Table 1Go). It is important to note that in Figs. 2–6GoGoGoGoGo, the amount of pro-TRH peptide (in picograms) is depicted for each gel, whereas in Tables 1Go and 2Go, the relative percentage of each proTRH peptide in femtomoles (which takes into account the molecular mass of each species) is depicted. Although approximately 28% and 25% of the total femtomole amount of immunoreactive peptides belonged to the pro-pEH24 and TRH-pEH24 forms in the PVN of CTR animals, respectively, only approximately 24% and 10% were measured under hypothyroid conditions, showing approximately 4% and 14% decreases for these peptides, respectively (P < 0.05 for both forms). In addition, the pEH24 peptide showed an increase of about 20% in the femtomole amount of proTRH in the PVN of PTU-treated animals compared with CTR animals. As judged by the amount of total picograms of peptide per total amount of protein, the biosynthesis of these peptides increased more than 2-fold in the PVN (Figs. 3AGo and 4AGo) consistent with up-regulation of the preproTRH gene. However, the values in the Lh (Figs. 3BGo and 4BGo) and POA (Figs. 3DGo and 4DGo) remained unaltered during PTU treatment.


Figure 4
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  		FIG. 4. Low iodine/PTU treatment altered the biosynthesis, release, and posttranslational processing of the N-terminal side of proTRH. This figure depicts an electrophoretic separation on a tricine-sodium dodecyl sulfate-polyacrylamide gel of PVN (A), Lh (B), ME (C), and POA (D) samples extracted from hypothyroid or CTR animals, followed by acid extraction of gel slices and RIA against pEH24 peptide. The molecular masses of the identified peaks are indicated based on the migration of standards and previously identified peaks using amino acid synthetic peptide standards (23 ). Each figure represents a typical profile of three independent experiments.

 

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  		TABLE 1. PTU and T4 treatments alter the ratio for each proTRH-derived peptide in the PVN

 

Figure 5
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  		FIG. 5. T4 treatment alters the normal biosynthesis and release of the C-terminal side of proTRH. This figure depicts an electrophoretic separation on a tricine-sodium dodecyl sulfate-polyacrylamide gel of PVN (A), Lh (B), ME (C), and POA (D) samples extracted from hyperthyroid or CTR animals, followed by acid extraction of gel slices and RIA against pYE17 peptide. The molecular masses of the identified peaks are indicated based on the migration of standards and previously identified peaks using amino acid synthetic peptide standards (23 ). Each figure represents a typical profile of three independent experiments.

 

Figure 6
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  		FIG. 6. T4 treatment altered the biosynthesis, release, and posttranslational processing of the N-terminal side of proTRH. This figure depicts an electrophoretic separation on a tricine-sodium dodecyl sulfate-polyacrylamide gel of PVN (A), Lh (B), ME (C), and POA (D) samples extracted from hypothyroid or CTR animals, followed by acid extraction of gel slices and RIA against pEH24 peptide. The molecular masses of the identified peaks are indicated based on the migration of standards and previously identified peaks using amino acid synthetic peptide standards (23 ). Each figure represents a typical profile of three independent experiments.

 

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  		TABLE 2. PTU and T4 treatments do not alter the ratio for each proTRH-derived peptide in the Lh

 
Hyperthyroidism induced changes in proTRH processing that resulted in an accumulation of proTRH-derived peptides in the PVN
In contrast to the results depicted in Fig. 2CGo, where the hyperthyroid condition showed a reduction in the biosynthesis of TRH, we observed an accumulation in the PVN of the 5.4-kDa C-terminal peptide as well as the N-terminal peptides (Figs. 5AGo and 6Go). However, a decrease in both peptides was seen in the ME (Figs. 5CGo and 6CGo). T4 treatment caused a significant increase inTRH-pHE24, the opposite of what was observed in hypothyroidism (Table 1Go). Similarly, less production of the end product pEH24 (–10%) was also observed after T4 treatment compared with its counterpart after PTU treatment (+20%). We found that the profiles of N- and C-terminal peptides were unchanged in the Lh and POA after T4 treatment (Fig. 5Go, B and D, and Fig. 6Go, B and D). Quantification of the percentages of femtomoles of pro-pEH24 and pEH24 in the Lh is shown in Table 2Go and reveals that thyroid status does not affect these peptides in the Lh.

Hypophysiotropic PC1/3, but not PC2, was affected by thyroid status, as determined by Western blot
Having demonstrated that changes in thyroid status alter the processing of proTRH, we determined the regulation of PC1/3 and PC2 by thyroid status. Figure 7AGo shows that in the PVN, there was an increase of approximately 20% in the PC1/3 level (87 kDa; P < 0.05) of PTU-treated animals and a decrease of approximately 10% (P < 0.05) in the PC1/3 level of T4-treated animals. Similar changes in PC1/3 levels were observed in the ME (Fig. 7BGo). In contrast, there were no significant differences in PC2 protein levels (75 and 68 kDa) in the PVN (Fig. 7CGo) or ME (Fig. 7DGo). The levels of PCs were unaffected in the Lh and POA regions outside the hypophysiotropic area (data not shown).


Figure 7
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  		FIG. 7. Effects of T4 and low iodine/PTU treatments on PC1 and PC2 proteins in the PVN and ME of rats. A and B, Typical Western blot for PC1/3 from PVN and ME in PTU-treated, CTR, and T4-treated animals. Sixty milligrams of total protein were loaded in each well for each condition. Below are shown the integrated ODs using National Institutes of Health Image software. C and D, Typical Western blot for PC2 from PVN and ME in PTU-treated, CTR, and T4-treated animals. Below are shown the integrated ODs using Image software. A total of six animals per condition was used in this study. ANOVA was followed by multiple comparison using the Tukey-Kramer test. *, P < 0.05, significant difference at this level. The molecular masses for PC1/3 and PC2 are indicated at the right of each gel.

 
Hypophysiotropic PC1/3 and PC2 were affected by thyroid status, as determined by immunohistochemistry
Due to the fact that removal of the entire PVN and ME regions (as in Fig. 7Go) also includes other neurons beside proTRH neurons, we used immunohistochemistry to examine PC1/3 and PC2 protein expression in specific regions of the hypothalamus. Using an anti-pYE17 antibody, Fig. 8Go demonstrates that proTRH staining neurons in the PVN were mainly expressed in the parvocellular region (Fig. 8AGo), which was increased in hypothyroid rats (Fig. 8BGo) and substantially reduced in hyperthyroid rats (Fig. 8CGo). Staining for PC1/3 was seen in paraventricular lateral magnocellular (PaLM), medial parvocellular (PaMP), and periventricular parvocellular cells (Fig. 8DGo) and was significantly increased in hypothyroid rats (Fig. 8EGo). In hyperthyroid rats, PC1/3-staining neurons were almost absent, with the exception of lower expression in PaLM (Fig. 8FGo). PC2-staining neurons were mainly expressed in the PaMP, with lower expression in the PaLM regions compared with PC1/3 expression (Fig. 8GGo). In hypothyroid rats (Fig. 8HGo), PC2 expression in the PVN was increased, whereas in hyperthyroid rats (Fig. 8IGo), reduced PC2 expression was seen. Figure 9Go demonstrates that hypothyroidism increased and hyperthyroidism decreased both the integrated intensity/area (integrated OD) of the positively staining neurons (A, C, and E) as well as the number of positively stained neurons/area (B, D, and F) for proTRH (A and D), PC1/3 (B and E), and PC2 (C and F) in the PVN. Thus, within the PVN region, proTRH, PC1/3, and PC2 expressions were responsive to thyroid status.


Figure 8
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  		FIG. 8. Immunohistochemical characterization of proTRH (A–C), PC1/3 (D–F), and PC2 (G–I) of the PVN of euthyroid (A, D, and G), hypothyroid (B, E, and H), and hyperthyroid (C, F, and I) rats. The anterior, mid, and caudal levels of the PVN are depicted. Scale bar, 50 µm. Arrows indicate immunopositive cells for TRH, PC1/3, and PC2.

 

Figure 9
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  		FIG. 9. Quantification of proTRH (A and D), PC1/3 (B and E), and PC2 (C and F) immunoreactivity in the PVN of euthyroid, hypothyroid, and hyperthyroid rats. Results are expressed as integrated intensity per area (integrated OD) of the positively staining neurons (A, C, and F) and as the number of positively stained neurons per area (B, D, and G). **, P < 0.05; ***, P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The regulation of plasma thyroid hormones is essential for maintenance of metabolic homeostasis and development in vertebrates. Normal homeostasis of the thyroid axis involves feedback at the level of the PVN by thyroid hormone, principally T3, to inhibit proTRH expression. The regulation of proTRH mRNA and TRH levels by thyroid status has been studied in rat hypothalamus, rat caudal nuclei, and rhesus monkey hypothalamus (27, 28, 29, 30). Low circulating levels of T4 and T3 increased preproTRH mRNA expression in the PVN, whereas increased levels of T4 and T3 suppressed preproTRH gene expression in the PVN (31, 32, 33). By in situ hybridization analysis, this response occurred exclusively in medial parvocellular neurons of the paraventricular nucleus (32, 33), suggesting that preproTRH mRNA levels in other regions of the hypothalamus were not regulated by T3. In the present study we demonstrated that the regulation of proTRH processing by thyroid status is a novel aspect in the regulation of the HPT axis. A low iodine/PTU diet, in addition to increasing hypophysiotropic preproTRH mRNA (32), increased the synthesis of proTRH-related peptides and pro-TRH processing, most likely by the actions of the PCs. Furthermore, proTRH neurons responsive to PTU were localized only in the medial and periventricular parvocellular division of the PVN, consistent with previous studies (34). The ME of hypothyroid rats was also found to have lower levels of N- and C-terminal proTRH-derived peptides compared with that of CTR rats. These results are consistent with previous studies suggesting that hypothyroidism increased the secretion of TRH and other proTRH-derived peptides from hypophysiotropic regions such as the ME, with no effect in nonhypophysiotropic areas such as the olfactory bulb (35).

Hyperthyroidism, in contrast, was found to decrease TRH in the PVN, consistent with a decrease in preproTRH mRNA and TRH staining in the PVN (36, 37). Our results indicate that the ME of hyperthyroid rats contained higher levels of TRH, suggesting a decrease in the release of TRH. Supporting these observations, previous studies by Rondeel et al. (38) showed that hypothalamic fragments of rats treated with T4 secreted 40% less TRH than hypothalami of euthyroid rats, and a similar decrease in secretion was observed in an in vivo model (38). In our experimental models, hypothyroid and hyperthyroid conditions produced contrary actions on the TRH in the ME and PVN of rats. Unexpectedly and differently from TRH, we found elevations of proTRH207–255 and pEH24-related peptide levels in the PVN of hyperthyroid rats. Although TRH levels increased in the ME of T4-treated rats, levels of the N- and C-terminal proTRH-derived peptides decreased.

Specific regulation of processing of proTRH is probably a key checkpoint where final amounts of biologically active peptides can be tightly regulated, as seen in this study and during opiate withdrawal (23), suckling (26), and fasting (24). We propose that the accumulation of intermediate forms of proTRH processing in hyperthyroidism was due to down-regulation of processing enzymes, PC1/3 and PC2. Analysis of proTRH processing in the PVN indicated that in hypothyroidism, the amounts of the precursor peptide (pro-pEH24) increased, and the processing products displayed a different pattern from that in CTR rats. In the PVN of hypothyroid rats, the increase in pEH24 was probably the result of increased processing of TRH-pEH24 to generate pEH24. Thus, under hypothyroid conditions, there is a selective increase in the biosynthesis of proTRH in the PVN associated with an enhanced posttranslational processing to increase the amount of proTRH-derived peptides, including pEH24 and TRH. In contrast, T4 treatment led to an accumulation of pro forms rather than more TRH biosynthesis, consistent with a down-regulation of processing enzymes. T4 treatment also produced alterations in posttranslational processing of proTRH in the PVN; there was decreased conversion of TRH-pEH24 into pEH24. In the ME of hyper- and hypothyroid rats, we did not detect the TRH-pEH24 extended form, suggesting that independently of changes in the PVN, the pEH24 peptide is already formed when it reaches the ME. Thus, these results suggest that hypothyroidism up-regulates and hyperthyroidism down-regulates preproTRH expression and its protein biosynthesis, which is coupled with the regulation of proTRH processing in a coordinated manner. However, in hypothyroidism, more TRH is released from the ME, leading to paradoxically lower levels of TRH remaining in the ME. The situation is reversed in hyperthyroidism, in that less TRH is released from the ME, leading to higher levels of TRH remaining in the ME.

The gene for proTRH has been studied, and the promoter was found to contain two negative thyroid hormone response elements (39, 40), by which thyroid hormone down-regulates preproTRH expression. The hypothalamic regions chosen in this study contain at least one kind of thyroid hormone receptor (41, 42) and should be susceptible to thyroid status. Our results suggest that coactivators or corepressors of preproTRH transcription differ in different brain regions. Moreover, coexpression of preproTRH mRNA together with both PCs mRNAs occurs in several of these regions (23, 43). Even though coexpression of preproTRH mRNA with both PC1/3 and PC2 mRNAs occurred abundantly in PVN and POA (10), our present results show that proTRH and PC expression in the Lh and POA was not affected by either hypo- or hyperthyroidism, consistent with results found by other investigators (31, 35, 44).

The Lh showed preproTRH mRNA coexpressing with PC2 mRNA, but not with PC1/3 mRNA (43), despite the high concentration of PC1/3 found in this region of the hypothalamus (10, 23, 45). PC2 is able to produce mature proTRH-derived peptides in the absence of PC1/3, as we demonstrated in PC1/3-null mice (unpublished observations). Because PC2 is active only in mature secretory vesicles, it is likely that in the Lh, the processing of proTRH commences later than that in the PVN or POA. In the Lh, we found that proTRH208–255 peptide was cleaved to generate a novel proTRH-derived peptide. The preproTRH sequence contains an Arg residue (a potential monobasic cleavage site) (46) at preproTRH233, and the antibody against the C-terminal side of proTRH used in this study recognized a small peptide of about 2.8 kDa that could match the proTRH234–255 sequence. Additional studies using mass spectrometry and microsequence analysis will determine the exact nature of this novel peptide. This differential processing in the Lh is in accordance with the concept that certain regions in the brain can give rise to different proTRH-derived peptides in addition to or instead of TRH.

In summary, our results support the hypothesis that hyperthyroidism down-regulates and hypothyroidism up-regulates PC expression and proTRH processing. This coupled regulation may lead to more effective processing of hypophysiotropic proTRH into mature peptides. The regulation of proTRH processing in the PVN by thyroid status is a novel and specific aspect of regulation of the HPT axis, which may have important implications in the overall physiology of this axis.


   Footnotes
 
This work was supported in part by National Institutes of Health Grants R01-DK-58148 (from the National Institute of Diabetes and Digestive and Kidney Diseases) and R01-NS-045231 (from National Institute of Neurological Disorders and Stroke; to E.A.N.) and Grant R01-DA-14659 (to T.C.F.).

The authors have nothing to declare.

First Published Online February 23, 2006

Abbreviations: CTR, Control; HPT, hypothalamic-pituitary-thyroid; Lh, lateral hypothalamus; ME, median eminence; PaLM, paraventricular lateral magnocellular; PaMP, medial parvocellular; PC, prohormone convertase; POA, preoptic area; PTU, propylthiouracil; PVN, paraventricular nucleus.

Received December 19, 2005.

Accepted for publication February 15, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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