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Am J Physiol Endocrinol Metab 292: E175-E186, 2007. First published August 22, 2006; doi:10.1152/ajpendo.00288.2006
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Cellular colocalization and coregulation between hypothalamic pro-TRH and prohormone convertases in hypothyroidism

Veronica Paez Espinosa,1 Monica Ferrini,2 Xiaoxiong Shen,1 Kabirullah Lutfy,1,3 Eduardo A. Nillni,4 and Theodore C. Friedman1

1Division of Endocrinology, Department of Medicine, Charles R. Drew University of Medicine and Sciences, University of California Los Angeles School of Medicine, Los Angeles; 2Division of Urology, Research and Education Institute, Harbor-University of California Los Angeles Medical Center, Torrance; 3College of Pharmacy, Western University of Health Sciences, Pomona, California; and 4Division of Endocrinology, Department of Medicine, Brown Medical School, Rhode Island Hospital, Providence, Rhode Island

Submitted 16 June 2006 ; accepted in final form 21 August 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The prohormone convertases (PCs), PC1/3 and PC2, are involved in the tissue-specific endoproteolytic posttranslational processing of many hormonal precursors within the secretory pathway. One important prohormone, pro-thyrotropin-releasing hormone (TRH), is expressed in both hypophysiotropic (where it regulates the secretion of thyroid-stimulating hormone) and nonhypophysiotropic regions of the brain. Pro-TRH is processed at specific sites in the secretory pathway, primarily by PC1/3 followed by PC2. We hypothesized that thyroid hormone status in specific nuclei of the brain would alter pro-TRH processing by inducing changes in PC1/3 and PC2 expression. Therefore, we examined pro-TRH, PC1/3, and PC2 coexpression and coregulation in the paraventricular nucleus (PVN), lateral hypothalamus (LH), and ventromedial nucleus (VMN) of hypothyroid and euthyroid rats. Our results show that 6-n-propyl-2-thiouracil (PTU) treatment producing hypothyroidism induced a significant increase in the expression of PC1/3, PC2, and pro-TRH in the PVN and LH, but not VMN. When confocal studies were performed, an increase in colocalization of PC1/3 or PC2 in pro-TRH was observed only in PVN, a response that was especially prominent in the ventral and medial areas of the PVN. PTU did not regulate colocalization in the VMH or LH. Regulation of colocalization of processing enzyme and prohormone expression is a novel mechanism to alter hormonal biosynthesis.

posttranslational processing; hyperthyroidism; hypothyroidism; prohormone convertase; hypothalamus; pro-thyrotropin-releasing hormone; peptide

PEPTIDE HORMONES AND NEUROPEPTIDES are often synthesized from larger inactive precursor proteins by endoproteolytic processing. The majority of prohormones are cleaved at paired basic residues to generate bioactive hormones in a cell-specific manner by prohormone convertases (PCs), members of the mammalian family of the subtilisin-like endoproteases (37, 43). Some peptide hormones need additional modifications, including further cleavage by exopeptidases, such as carboxypeptidase E (CPE) or carboxypeptidase D (CPD), to remove the carboxy-terminal basic amino acids (5, 7), acetylation at the amino terminus by N-acetyltransferases, or by amidation at their carboxyl terminus by the action of peptidylglycine {alpha}-amidating monooxygenase enzyme (6, 42). Although seven members of the PC family have been cloned, PC1/31 and PC2 are found exclusively in neural and endocrine cells equipped with a regulatory secretory pathway (4, 11, 38, 39). PC1/3 and PC2 process a variety of prohormones and proneuropeptides, including pro-thyrotropin-releasing hormone (TRH), proopiomelanocortin, prosomatostatin, provasopressin, proinsulin, and pro-islet amyloid polypeptide (1, 3, 8–10, 44). Our laboratories have used biochemical techniques to understand the initial posttranslational modifications of the pro-TRH precursor. We found that the initial cleavages at paired basic residues were primarily by PC1/3 and secondarily by PC2 (9, 25, 26, 35). The intermediate products of pro-TRH generated from these enzymatic cleavages are further modified by exopeptidases, such as CPE or CPD, to remove the carboxy-terminal basic amino acids (28) and then amidation at their carboxyl terminus by the action of peptidylglycine {alpha}-amidating monooxygenase enzyme (6) to generate TRH (pGlu-His-Pro-NH2; thyroliberin).

Both PC1/3 and PC2 are widely distributed in the brain, with higher levels of PC2 in most regions (34). Although studies on the regulation of PCs have lagged behind biochemical characterization, our laboratories have shown that the PCs are regulated by states of hyperglycemia (23), inflammation (20), sucking (24), starvation (32) and exposure to opioids (22). In our studies using rats exposed to streptozotocin, we found that the diabetic state led to altered {alpha}-cell processing of proglucagon to give increased levels of glucagon-like peptide 1 (23). We have also localized regions on PC1/3 and PC2 human promoters that contain putative negative thyroid hormone response elements and have also shown that triiodothyronine (T3) negatively regulates PC1/3 and/or PC2 expression in rat GH3 cells, rat anterior pituitary, hypothalamus, and cerebral cortex (18, 19, 40, 41). It has been assumed that regulation of both prohormone and PC within the same tissue would lead to altered hormonal biosynthesis; however, this assumption requires that the changes in prohormone and PC levels occur in the same cell, an assumption that has not been proven. It is possible that different activators or repressors acting on the promoter of either the prohormone or the PC in different cell types would lead to cell-specific regulation so that coregulation does not occur.

TRH plays an important role in the hypothalamic-pituitary-thyroid (HPT) axis. TRH, synthesized as pro-TRH and processed in the paraventricular nucleus (PVN; primarily in the parvocellular division) of the hypothalamus stimulates the biosynthesis and secretion of thyrotropin (TSH) from the pituitary (12, 13). TSH, in turn, stimulates thyroxine (T4) and T3 biosynthesis and release from the thyroid. TRH neurons in the PVN project to the median eminence, where they are in close proximity to the capillaries of the hypophysial-portal system. Hence, TRH originating in the PVN is referred to as hypophysiotrophic TRH. The maintenance of euthyroidism is dependent on a highly regulated balance of positive and negative feedback, in which TRH positively regulates TSH and thyroid hormones suppress pro-TRH expression and TSH secretion. Pro-TRH is also abundantly expressed in hypothalamic neurons outside of the PVN and in extrahypothalamic brain regions. This extrahypophysiotropic TRH is believed to function as a neuromodulator of neurotransmitters (21, 47). Extrahypophysiotropic TRH has a role in appetite control, arousal and sleep, cognition, locomotion antinociception, thermoregulation, and psychological function (27). Little is known about the regulation of extrahypophysiotropic TRH by thyroid hormone status.

In our recently published paper (29) using biochemical techniques, we demonstrated that alteration in thyroid status only affected pro-TRH processing in the PVN and did not affect processing of extrahypophysiotropic pro-TRH. We found that hypothyroidism increased both PC1/3 and PC2 and pro-TRH in the PVN, whereas hyperthyroidism did not affect PC1/3 and PC2 and pro-TRH expression. Accumulation of a carboxy-terminal 5.4-kDa pro-TRH peptide was seen, presumably because of altered secretion. Because the effects of hypothyroidism on PC1/3 and PC2 and pro-TRH expression were profound, we used immnofluorescence techniques to study the effects of hypothyroidism on the cellular colocalization and coregulation of pro-TRH, PC1/3, and PC2 expression in the PVN and selected extrahypophysiotropic sites known to express pro-TRH [lateral hypothalamus (LH) and ventromedial nucleus (VMN)]. We expected that alterations in thyroid status would change substrate (pro-TRH) and processing enzyme (PC1/3 or PC2) in parallel and in the same cell, leading to altered processing of pro-TRH to active peptides. Surprisingly, our current paper demonstrates that 6-n-propyl-2-thiouracil (PTU)-induced hypothyroidism increased pro-TRH, PC1/3, and PC2 protein expression in both hypophysiotropic and extrahypophysiotropic regions, yet the increase in PC1/3 and PC2 in pro-TRH neurons (increased colocalization) only occurred in the PVN. Thus regulation of prohormone and enzyme may (in the PVN) or may not (extrahypophysiotropic regions) lead to altered hormonal biosynthesis. The selective coregulation of pro-TRH processing by thyroid status in the PVN is a novel aspect of the regulation of the HPT axis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and treatments. All experimental protocols and animal procedures were approved by the Institutional Animal Care and Use Committee of Charles R. Drew University of Medicine and Sciences-University of California Los Angeles School of Medicine and performed in compliance with the National Institutes of Health Guidelines for the Use of Animals in Research. Adult male Sprague-Dawley rats (Harlan, Indianapolis, IN) weighing 150–180 g were housed in a room with controlled light, temperature, and humidity. Two groups of eight animals each were treated as follows. The first group (control) was allowed free access to standard rodent chow and water. The second group (hypothyroid) received rodent chow containing low iodine plus 0.15% PTU (Harlan-Teklad, Madison, WI) and water ad libitum. This protocol of PTU treatment consistently resulted in low serum T4 levels and elevated serum TSH levels in the PTU-treated group, as described in our prior manuscript (29). Animals had free access to food and water and were killed after 21 days of treatment.

Immunofluorescence. For immunohistochemical experiments, both groups of animals were anesthetized with pentobarbital sodium (45 mg/kg body wt ip) and used 10–15 min later, i.e., as soon as anesthesia was assured by the loss of pedal and corneal reflexes. Both groups of rats were perfused with PBS (pH 7.2), followed by 4% paraformaldehyde in PBS. Brains were carefully removed, postfixed in 4% paraformaldehyde overnight at 4°C, immersed in 25% sucrose until they sunk, and then frozen in dry ice. Eight-micrometer-thick coronal cryostat sections were cut mounted on slides (Superfrost/Plus; Fisher-Scientific, Pittsburg, PA) and processed for immunohistochemistry. Antigen retrieval was performed by heating the slides in a microwave oven with Antigen Unmasking Solution (Vector, Burlingame, CA) for three times of 1 min each and then cooled for 20 min. For detection of the first primary antibodies, PC1/3 and PC2, a Biotin-Conjugated Tyramide Signal Amplification kit (TSA) (Perkin-Elmer Life Sciences, Boston, MA) was used. Because tyramide deposition in the immediate neighborhood of the antigen is catalyzed by the presence of horseradish peroxidase (HRP), endogenous peroxidase activity of frozen sections was blocked by placing the slides in 0.3% H2O2 for 20 min.

For staining with PC1/3 and PC2 primary antibodies, brain slices were incubated with TNB blocking buffer [0.1 M Tris (pH 7.5), 0.15 M NaCl, and 0.5% blocking reagent (TSA kit)] for 40 min and then incubated with PC1/3 or PC2 polyclonal antibodies (1:400) for 16 h at 4°C. These antibodies were raised against either a PC1/3-glutathione fusion protein or a PC2-glutathione fusion protein (14) and generously provided by Dr. Nigel Birch (University of Auckland). After being rinsed with PBS, sections were incubated with Biotin-SP-conjugated AffiniPure Fab Fragment Donkey Anti-Rabbit IgG (H + L; Jackson ImmunoResearch, West Grove, PA) at a 1:400 dilution in 0.5% blocking reagent (TSA kit) for 60 min, followed by streptavidin-HRP (TSA kit) 1:100 for 30 min, and finally incubated in Fluorophore Tyramide Amplification Reagent (supplied in TSA kit) at a dilution of 1:50 for 10 min.

For staining with pro-TRH primary antibody, brain slices were incubated with PBS-0.3% Triton X-100 [PBS-T 0.3% (pH 7.2)] plus 1% normal donkey serum (NDS; Jackson ImmunoResearch) for 40 min. Slides were then incubated for 16 h at 4°C with pro-TRH antibody pYE17 (1:25,000), which recognizes both pro-TRH and carboxy-terminal intermediate products (26). At the end of the incubation period, slides were rinsed three times for 5 min each and incubated with rhodamine [Rhodamine Red AffiniPure Fab Fragment Donkey Anti-rabbit IgG (H + L), 1:200; Jackson ImmunoResearch] in 1% NDS for 1 h at room temperature.

Unless indicated otherwise, all slides were rinsed three times for 5 min in 0.1 M Tris, pH 7.5, 0.15 M NaCl, and 0.05% Tween 20 after each incubation. All incubations except with the primary antibody were carried out at room temperature in a humidified container and protected from light.

Double immunofluorescence. Before the addition of the second primary antibody, the already labeled PC1/3- and PC2-stained sections were rinsed and incubated in PBS-T 0.3% (PBS, pH 7.2, containing 0.3% Triton X-100) plus 1% NDS for 40 min. Slides were then incubated for 16 h at 4°C with pro-TRH antibody pYE17 (1:25,000), which recognizes both pro-TRH and carboxy-terminal intermediate products (26). At the end of the incubation period, slides were rinsed three times for 5 min each and incubated with rhodamine [Rhodamine Red AffiniPure Fab Fragment Donkey Anti-rabbit IgG (H + L); Jackson ImmunoResearch] 1:200 for 1 h at room temperature. Rinsed slides were mounted using Vectashield mounting medium for fluorescence with DAPI (Vector Laboratories). Negative controls for the double-immunofluorescence procedure were performed by substitution of nonimmune serum for the primary or secondary antisera. For the TSA-enhanced procedure, staining controls were performed by omission of the primary antiserum for either the first or second incubation, or both. A nonamplified staining with antibodies against PC1/3 and PC2, using the same concentration (1:400) as for the TSA-enhanced reaction, was routinely included.

Immunostained sections were analyzed using fluorescence microscopy (Leica Microsystems, Bannockburn, IL). Images were captured with a SPOT RT (Diagnostic Instruments, Sterling Heights, MI) color digital camera attached to the microscope, and the images captured were saved in JPEG files. Quantification of protein expression for pro-TRH, PC1/3, and PC2 was determined by calculating the integrated optical density (IOD) of immunostained fields with each corresponding antibody using Image Pro Plus software (Media Cybernetics, Silver Spring, MD). Using this software, IOD results are proportional to the mean optical density per area, which reflects the concentration of immunoreactive antigen. Two anatomically matched adjacent brain sections from each animal were averaged, and, from each section, we analyzed approximately four images at x200. Eight images per animal were analyzed, and this value was averaged with another three animals per group. DAPI staining (nucleus) was used to recognize the location of cells with their adjacent cytoplasmic staining, and we found that the number of DAPI-stained cells/field were not affected by treatment. A second group of four rats in each group was also analyzed to confirm the results in the first group and, at times, to obtain better quality pictures, but were not included in the quantitation.

To assure enzyme/proprotein colocalization, confocal laser-scanning microscopy (Leica TST1200 at the Gunther core facility at Harbor-UCLA Research and Education Institute, Torrance, CA) equipped with argon and He Ne lasers coupled to acquisition software was used. The images were saved as TIFF files, imported to Adobe Photodeluxe 1.0, cropped, and adjusted for brightness and contrast only.

The percent of colocalization of PC1/3 and TRH was determined by counting the number of yellow-stained cells (colocalized) over the total number of cells per area stained with DAPI in the entire image that comprises the nucleus or brain region of interest and expressed as a percentage.

Statistical analyses. Data are expressed as means ± SE. One-way ANOVA with Dunnett’s post hoc t-test was used. Significance was at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
PC1/3, PC2, and pro-TRH immunoreactivity in the PVN of hypothyroid rats. We performed single immunofluorescence against PC1/3, PC2, and pro-TRH proteins and found that neurons in the PVN of euthyroid animals showed discrete expression of PC1/3 and PC2 proteins (Fig. 1, A and B) as well as pro-TRH peptide (Fig. 1C). PC1/3 neurons in the PVN of euthyroid animals were mainly expressed in the paraventricular hypothalamic nucleus, ventral area (PaV; Fig. 1, A and J). There was lower expression in the paraventricular hypothalamic medial area (PaMP; Fig. 1, A and J; the PaV and the PaMP make up the parvocellular neurons of the PVN) and almost no expression in the lateral region of the paraventricular nucleus that comprises the magnocellular neurons. PC2-stained neurons showed slightly less expression than PC1/3 neurons (Fig. 1B) but followed the same pattern of distribution to that of PC1/3 in euthyroid rats (Fig. 1J). Pro-TRH neurons were present in PVN of control rats (Fig. 1C), and the few pro-TRH-stained neurons observed were located exclusively in the PaV region (Fig. 1J).


Figure 1
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  		Fig. 1. Immunofluorescence characterization (A–F) and quantification (G–I) of positively stained neurons for prohormone convertase (PC) 1/3 (green fluorescence in A, D, and G), PC2 (green fluorescence in B, E, and H), and pro-thyrotropin-releasing hormone (TRH; rhodamine in C, F, and I) in the paraventricular nucleus (PVN) of euthyroid (A–C) and hypothyroid (D–F) rats. Quantification of PC1/3 (G), PC2 (H), and pro-TRH (I) immunoreactivity is expressed as integrated optical density/area. *P < 0.05 and **P < 0.01. The anterior, middle, and caudal levels of the PVN are depicted with a schematic representation of the staining (black dots) in normal (J) and hypothyroid (K) rats. Both the ventral PVN (PaV; aqua) and medial PVN (PaMP, purple) are part of the parvocellular neurons, as depicted in J and K. PaPo, PVN, posterior; 3V, 3rd ventricle; Pe, periventricular area; f, fornix. Scale bar = 50 µm.

 
PC1/3 and PC2 protein expression significantly increased in PVN of hypothyroid rats (Fig. 1, D, E, G, and H). This response was found to be selective in the PaV and PaMP areas of the PVN and was parallel to the increase observed in the PVN of pro-TRH-stained neurons of hypothyroid rats (Fig. 1, F and K).

PC1/3, PC2, and pro-TRH expression in rat LH and VMN in hypothyroid rats. PC1/3 (Fig. 2A), PC2 (Fig. 2B), and pro-TRH (Fig. 2C) neurons were also expressed in the LH nucleus (we examined a more dorsal portion of the LH) of euthyroid rats (Fig. 2J). When compared with their respective controls (Fig. 2, A, B, C, and J), hypothyroid animals showed a significant (P < 0.05) increase in the number of PC1/3 (Fig. 2, D and G), PC2 (Fig. 2, E and H), and pro-TRH (Fig. 2, F and I) positive neurons but did not show any change in the pattern of distribution inside the nucleus (Fig. 2K).


Figure 2
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  		Fig. 2. Immunofluorescence characterization (A–F) and quantification (G–I) of the distribution of positive staining of PC1/3 (green fluorescence in A, D, and G), PC2 (green fluorescence in B, E, and H), and pro-TRH (rhodamine in C, F, and I) in the lateral hypothalamus (LH) of euthyroid (A–C) and hypothyroid (D–F) rats. Quantification of PC1/3 (G), PC2 (H), and pro-TRH (I) immunoreactivity is expressed as integrated optical density/area. *P < 0.05 and **P < 0.01. Schematic representations of the staining (black dots) for normal (J) or hypothyroid (K) rats are shown. DMN, dorsomedial nucleus. Scale bar = 50 µm.

 
In the VMN of control rats, we found discrete immunoreactivity for PC1/3 (Fig. 3A), PC2 (Fig. 3B), and pro-TRH (Fig. 3C). PC1/3 and PC2 positive neurons were distributed mainly in the dorsal (VMND) and central (VMNC) region of the VMN, with only few numbers of positive neurons located at the ventral part (VMNV) of the nucleus. The number of PC1/3 positive neurons was slightly higher than those of PC2 (Fig. 3, J and K). Neurons of the VMN of euthyroid rats also showed immunoreactivity to pro-TRH antibodies restricted to the dorsal and central portions of the nucleus (Fig. 3C). The VMN of hypothyroid rats displayed the same characteristics in number and distribution of PC1/3 (Fig. 3, D and G), PC2 (Fig. 3, E and H), and pro-TRH (Fig. 3, F and I) as those described for euthyroid animals. Thus there is regulation by thyroid status of the expression of PC1/3, PC2, and pro-TRH neurons in the LH but not the VMN regions.


Figure 3
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  		Fig. 3. Immunofluorescence characterization (A–F) and quantification (G–I) of stained neurons for PC1/3 (green fluorescence in A, D, and G), PC2 (green fluorescence in B, E, and H) and pro-TRH (rhodamine in C, F, and I) in the ventromedial nucleus of euthyroid (A–C) and hypothyroid (D–F) rats. Quantification of PC1/3 (G), PC2 (H), and pro-TRH (I) immunoreactivity is expressed as integrated optical density/area. The anterior, middle, and caudal levels of the ventromedial nucleus are depicted with a schematic representation of the typical staining observed for PC1/3 (J, black dots), PC2 (K, black dots), and pro-TRH (L, black dots) in both control and 6-n-propyl-2-thiouracil (PTU)-treated rats. VMND, ventromedial nucleus, dorsal; VMNC, ventromedial nucleus, central; VMNV: ventromedial nucleus, ventral; DMD, dorsomedial nucleus, dorsal; MEI, medium eminence. Scale bar = 50 µm.

 
Colocalization of PC1/3 and PC2 positive neurons with pro-TRH staining neurons. To determine the presence of colocalized pro-TRH and PC1/3 or pro-TRH and PC2 neurons in the hypothalamic nuclei already described, we used confocal microscopy. Double staining in the PVN of euthyroid rats showed partial colocalization between PC1/3 and pro-TRH (Fig. 4A) or between PC2 and pro-TRH (data not shown). Interestingly, parallel to the significant increase in the number of stained neurons in the PVN of hypothyroid rats, there was also a significant increase in the amount of double staining between PC1/3 and pro-TRH (Fig. 4, B and C). This increase in the colocalization was observed also between PC2- and pro-TRH-stained neurons (Fig. 4D) and was located in the PaV and PaMP.


Figure 4
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  		Fig. 4. Confocal microscopy of neurons immunoreactive to pro-TRH and PC1/3 in the PaV region of the PVN in euthyroid (A) and hypothyroid (B) rats. Immunodetection was performed with PC1/3 primary antiserum followed by FITC biotinylated secondary anti-rabbit goat IgG (green staining). The stained slices were then treated with pro-TRH primary antibody followed by Rhodamine Red AffiniPure Fab Fragment Donkey Anti-rabbit IgG (H + L; red staining) secondary antibody. Yellow staining denotes overlay between pro-TRH and PC1/3 in euthyroid rats (A) and hypothyroid rats (B). C: colocalization of pro-TRH with PC1/3. D: colocalization between pro-TRH and PC2 in the PVN of euthyroid and hypothyroid rats. Results are expressed as %PC1/3 (labeled as PC1 for clarity) or PC2 reactive neurons that colocalize with pro-TRH positive neurons. Data are means ± SE. **P < 0.001. Magnification = x160 (left) and x400 (right). Scale bar = 40 µm (left) and 16 µm (right).

 
Similar to that observed in the PVN, double staining of the LH showed a moderate (~40%) level of neuronal colocalization of PC1/3 and pro-TRH (Fig. 5, A-C) or colocalization of PC2 and pro-TRH (data not shown) in control rats. Hypothyroidism induced an increase in the number of stained cells but, importantly, not an increase of colocalization between PC1/3 and pro-TRH (Fig. 5, D-G) or PC2 and pro-TRH (Fig. 5H).


Figure 5
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  		Fig. 5. Confocal microscopy of neurons immunoreactive to pro-TRH and PC1/3 in the LH of euthyroid [PC1/3 (A), pro-TRH (B), and colocalization (C)] and hypothyroid [PC1/3 (D), pro-TRH (E), and colocalization (F)] rats. Double staining was performed with PC1/3 primary antiserum followed by FITC biotinylated secondary anti-rabbit goat IgG (green staining). The stained slices were then treated with pro-TRH primary antibody followed by Rhodamine Red AffiniPure Fab Fragment Donkey Anti-rabbit IgG (H + L; red staining) secondary antibody. Immunodetection was performed by confocal microscopy. Yellow staining (white arrows) denotes overlay between pro-TRH and PC1/3 in euthyroid rats (C) and hypothyroid rats (F). Green arrows show noncolocalized neurons. G and H: colocalization of pro-TRH with PC1/3 (G) and pro-TRH with PC2 (H) in the LH of euthyroid and hypothyroid rats. Results are expressed as %PC1/3 or PC2 reactive neurons that colocalize with pro-TRH positive neurons. Magnification = x400. Scale bar = 16 µm.

 
In the VMN regions, PC1/3 (Fig. 6A) and pro-TRH (Fig. 6C) or PC2 (Fig. 6B) and pro-TRH (Fig. 6D) colocalized in the dorsal and central regions (Fig. 6, E and F). This colocalization was similar in both the normal (data not shown) and hypothyroid (Fig. 6, E and F) rats. Although the distribution of PC1/3 (Fig. 7A) and PC2 (Fig. 7B) positive neurons differs from that of pro-TRH at the level of VMNV (Fig. 7C), both VMND and VMNC regions show a high degree of colocalization in euthyroid rats (data not shown), and this pattern applied to the VMN of hypothyroid animals (Fig. 7, D and E). This lack of change in the coexpression between PC1/3 or PC2 and pro-TRH following hypothyroidism shows that, in the VMN, neither PC1/3 nor PC2 in pro-TRH neurons is coregulated by hypothyroid status.


Figure 6
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  		Fig. 6. Confocal microscopy of neurons immunoreactive to pro-TRH and PC1/3 or pro-TRH and PC2 in the ventromedial nucleus (VMN) of hypothyroid rats. Immunodetection was performed with PC1/3 or PC2 primary antiserum followed by FITC biotinylated secondary anti-rabbit goat IgG. The stained slices were treated with pro-TRH primary antibody followed by Rhodamine Red AffiniPure Fab Fragment Donkey Anti-rabbit IgG (H + L; red staining) secondary antibody and analyzed using confocal microscopy. Yellow staining denotes overlay between pro-TRH and PC1/3 or PC2 in hypothyroid rats. Magnification = x400. Scale bar = 16 µm.

 

Figure 7
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  		Fig. 7. Distribution of PC1/3, PC2, and pro-TRH positive neurons in the VMN of the hypothalamus of hypothyroid rats. The anterior, middle, and caudal levels of the VMN are depicted with a schematic representation of PC1/3 (A), PC2 (B), and pro-TRH (C) positive neurons. D and E: colocalization between pro-TRH and PC1/3 (D) and pro-TRH with PC2 (E) in each area of the VMN of euthyroid vs. hypothyroid rats. Results are expressed as %PC1/3 or PC2 reactive neurons that colocalize with pro-TRH positive neurons.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The HPT axis is tightly regulated at all levels of the axis. The cloning of the pro-TRH gene in 1986 (17) allowed for the studies showing that thyroid status inversely regulated pro-TRH mRNA and protein specifically in the PVN, with hypothyroidism causing increased expression that was reversed by T4 or T3 treatment (16, 36). Segerson et al. (36) showed that the regulation of pro-TRH expression by thyroid status was specific for the medial parvocellular region of the PVN, although hypothyroidism was found to regulate pro-TRH mRNA in the medullary caudal raphe nuclei as well (46). Surprisingly, Yamada and Wilber (45) found that TRH content in hypothalamic extracts was decreased in hypothyroidism, a paradox resolved by finding that hypothyroidism increased secretion of TRH and processed pro-TRH peptides from hypothalamic extracts (2). Thus hypothyroidism leads to increased paraventricular pro-TRH mRNA and pro-TRH intermediates, decreased paraventricular TRH, and increased TRH secretion, whereas hyperthyroidism leads to decreased pro-TRH mRNA, pro-TRH intermediates, and paraventricular TRH.

Our results agree with those of Koller et al. (16) and Segerson et al. (36) in that we found that hypothyroidism regulated pro-TRH expression most noticeably in the medial paraventricular nucleus. Our results differ from these groups in that we found that hypothyroidism regulated pro-TRH expression in the LH but not in the VMN. Koller et al. (16), using in situ hybridization, only examined PVN and thalamic pro-TRH mRNA expression and found that only PVN expression was regulated by thyroid status. Segerson et al. (36) depicted pro-TRH mRNA expression regulated by hypothyroidism in the PVN and LH and concluded that there were changes only in the PVN. However, inspection of the figure does seem to indicate that increased pro-TRH expression was also found in the LH in hypothyroidism, although statistical analysis was not given.

For processing to occur, both precursor and processing enzyme need to be in the same cell and in the same cellular compartments. It is thought that coordinated regulation between prohormone and PC occurs (1, 20); however, we demonstrated that regulation of {alpha}-cell PC1/3 occurred independent of regulation of proglucagon mRNA in a streptozotocin-induced diabetic model (23). Pu et al. (30) reported coexpression of pro-TRH with PC1/3 or PC2 mRNA in several brain regions, finding differential colocalization of PC1/3 or PC2 mRNA with TRH mRNA. Some PVN TRHergic neurons contained either PC1/3 or PC2 mRNA, whereas PC1/3 or PC2 mRNA was not present in thalamic reticular nucleus. Sanchez et al. (31) also confirmed PC1/3 and PC2 expression in pro-TRH neurons and found that PC2 mRNA was present in 60–70% of pro-TRH neurons and PC1/3 mRNA in 37–46% of pro-TRH neurons. Colocalization of pro-TRH with PC1/3 and PC2 in primary cultures of hypothalamic neurons was also shown by Schaner et al. (35).

Our results suggest that hypothyroidism selectively induces the expression of PCs in pro-TRH neurons of the PVN as a compensatory mechanism to increase the production of TRH and, eventually, thyroid hormone and that this mechanism is specific of the neurons located in the PaV and PaMP regions of the PVN. Most interestingly, although thyroid status regulates pro-TRH and PC1/3 and PC2 in the LH, coregulation does not occur in this region, and the amount of TRH peptide is not changed as we have previously described using biochemical techniques (29). In the VMN regions of the hypothalamus, neither expression nor coexpression was regulated by thyroid status.

Our study is the first to examine the cellular localization of coregulation of prohormone and PCs. Using conditions that render a rodent to be hypothyroid, we found that pro-TRH, PC1/3, and PC2 protein increase with hypothyroidism in the PVN and LH but not VMN regions of the hypothalamus. Most importantly, hypothyroidism induced a selective increase in the colocalization of PC1/3 and PC2 in pro-TRH neurons only in the PVN. Under euthyroid conditions, ~35% of pro-TRH neurons in the PVN expressed PC1/3 and 20% expressed PC2. Following hypothyroidism, close to 90% of the pro-TRH neurons expressed PC1/3 and 75% of the pro-TRH neurons expressed PC2 (Fig. 4). This increase in coexpression was selective, since it did not occur in the LH or VMN region.

What are the molecular mechanisms underlying this selective increase in colocalization? The gene for pro-TRH has been studied, and the promoter was found to contain two negative thyroid hormone response elements (nTREs; see Refs. 15 and 33) by which thyroid hormone downregulates pro-TRH expression. We have previously characterized the negative TRH on both the PC1/3 and PC2 human promoters that contain putative nTRE 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, 40, 41). Interestingly, the regulation was not uniform throughout the brain; for example, PC2 was not regulated by thyroid status in the hippocampus. We postulated that differential expression of TRH coactivators and corepressors in different regions is responsible for this selective regulation. Our study further expands this idea and suggests that not only are TRH coactivators and corepressors found in different regions, but they are present in different neurons in the same brain region and account for the selective increase in colocalization of PC1/3 and PC2 in pro-TRH neurons of the PVN. We propose to study the factors that interact with the promoters of PC1/3 and PC2 to give specific coexpression in pro-TRH neurons.

In contrast to hypothyroidism, which regulates pro-TRH, PC1/3, and PC2 in the PVN by both immunofluorescence techniques (this study) and biochemical techniques (29), we previously demonstrated that hyperthyroidism did not regulate hypothalamic pro-TRH, PC1/3, and PC2 expression (29). It is possible that in hyperthyroidism, other mechanisms, such as more profound regulation of TSH at the level of the pituitary, may be involved in regulating the HPT axis.

In conclusion, our data describe that hypothyroidism causes upregulation of PC1/3 and PC2 expression only in pro-TRH neurons of the PVN. As depicted in our recent paper (29), this leads to increased pro-TRH processing and increased TRH content in the PVN of hypothyroid animals. This is accompanied by increased TRH release from the median eminence and decreased TRH content in this region. It is noteworthy that the current paper showed that, although both pro-TRH and PC1/3 and PC2 expression are increased in the LH of hypothyroid animals, TRH peptide levels are not changed in that region (29) because the increase in PC1/3 and PC2 did not occur in pro-TRH neurons. Thus increases in TRH biosynthesis in hypothyroidism only occur in the PVN because the increase in colocalization of pro-TRH and PC1/3 as well as PC2 only occurred in those TRHergic neurons.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
T. C. Friedman and X. Shen are supported by National Institutes of Health (NIH) Center of Clinical Research Excellence Grant U54 RR-14616-01 to Charles R. Drew University of Medicine and Sciences. This work was also supported by Thyroid Research Advisory Council (Knoll Pharmaceutical Company) Grant SYN-0400-02, NIH Grant R01 DA-14659 to T. C. Friedman, RCMI Program Grant G21 RR-03026-13, and NIH Minority Institution Drug Abuse Research Program Grant R24DA-017298 to Charles R. Drew University of Medicine and Sciences. K. Lutfy was supported by NIH Grant R01 DA-16682. E. Nillni was supported by NIH Grants R01 DK-58148 and R01 NS-045231.


   ACKNOWLEDGMENTS
 
Current address for X. Shen: South Baylo University, Medical Research Center, 1126 N. Brookhurst St., Anaheim, CA 92801.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. C. Friedman, Charles R. Drew Univ. of Medicine & Sciences, Division of Endocrinology, 1731 E. 120th St., Los Angeles, CA 90059 (e-mail: tefriedm{at}cdrewu.edu)

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.

1 At the 6th Gordon Research Conference on Proprotein Processing, Trafficking and Secretion (2004), the leading researchers agreed to use the terminology PC1/3 to describe the identical PC1 and PC3 prohormone convertase. Back


   REFERENCES
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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