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Endocrinology, doi:10.1210/en.2007-0522
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Endocrinology Vol. 148, No. 10 4952-4964
Copyright © 2007 by The Endocrine Society

Cold Exposure Increases the Biosynthesis and Proteolytic Processing of Prothyrotropin-Releasing Hormone in the Hypothalamic Paraventricular Nucleus via ß-Adrenoreceptors

Mario Perello, Ronald C. Stuart, Charles A. Vaslet and Eduardo A. Nillni

Division of Endocrinology (M.P., R.C.S., C.A.V., E.A.N.), Department of Medicine, Brown University/Rhode Island Hospital, Department of Molecular Biology, Cell Biology, and Biochemistry (E.A.N.), and Department of Pathology and Laboratory Medicine (C.A.V.), Brown University, 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, Third Floor, Room 320, Providence, Rhode Island 02903. E-mail: eduardo_nillni{at}brown.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Different physiological conditions affect the biosynthesis and processing of hypophysiotropic proTRH in the hypothalamic paraventricular nucleus, and consequently the output of TRH. Early studies suggest that norepinephrine (NE) mediates the cold-induced activation of the hypothalamic-pituitary-thyroid axis at a central level. However, the specific role of NE on the biosynthesis and processing of proTRH has not been fully investigated. In this study, we found that NE affects gene transcription, protein biosynthesis, and secretion in TRH neurons in vitro; these changes were coupled with an up-regulation of prohormone convertase enzymes (PC) 1/3 and PC2. In vivo, NE is the main mediator of the cold-induced activation of the hypothalamic-pituitary-thyroid axis at the hypothalamic level, in which it potently stimulates the biosynthesis and proteolytic processing of proTRH through a coordinated up-regulation of the PCs. This activation occurs via ß-adrenoreceptors and phosphorylated cAMP response element binding signaling. In contrast, {alpha}-adrenoreceptors regulate TRH secretion but not proTRH biosynthesis and processing. Therefore, this study provides novel information on the molecular mechanisms of control of hypophysiotropic TRH biosynthesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MOST NEUROPEPTIDE HORMONES are synthesized from larger inactive precursors (1, 2, 3, 4). TRH is a key neuropeptide derived from proTRH by posttranslational processing (2). ProTRH is primarily cleaved by the prohormone convertase (PC) 1/3, although PC2 also cleaves some of the intermediate proTRH peptides (5, 6). Products of these enzymatic cleavages are subjected to the removal of C-terminal basic amino acids mainly by carboxypeptidase (CP) E and CPD (7). Finally, peptidylglycine {alpha}-amidating monooxygenase enzyme (PAM) amidates the carboxyl terminus of the immediate TRH progenitor, Gln-His-Pro-Gly (TRH-Gly), and the Gln residue undergoes cyclization to a pGlu residue to yield TRH (2). All these steps are essential for the generation of bioactive TRH.

TRH neurons located in the hypothalamic paraventricular nucleus (PVN) control hypothalamic-pituitary-thyroid (HPT) axis activity via release of TRH to the median eminence (ME) (2). In the pituitary, TRH stimulates secretion of TSH that, in turn, stimulates the release of thyroid hormones (2). Thyroid hormones are key stimulators of energy expenditure through the activation of mitochondrial uncoupling proteins in peripheral tissues (8). Previous studies showed that cold exposure activates the HPT axis at the hypothalamic level by increasing preproTRH mRNA in the PVN (9, 10, 11). This activation is vital as indicated by the fact that hypothyroid rats do not survive when they are exposed to cold (8). The neurotransmitter norepinephrine (NE) has been implicated as an important mediator of this activation (12, 13). However, the specific role of NE on the biosynthesis of TRH in the hypothalamic PVN has not been fully studied. In addition, we recently showed that TRH synthesis is regulated at the posttranslational level (14, 15, 16). Changes in thyroid, leptin, and melanocortin system status potently alter proTRH biosynthesis in concert with the PC1/3 and PC2 enzymes (14, 15, 16). However, whether NE can regulate processing enzymes is completely unknown. In the present study, we investigated the role of NE in the biosynthesis and posttranslational processing of TRH. Additionally, we determined the role of NE in the cold-induced activation of the HPT axis, the specific adrenoreceptor, and the intracellular signaling that mediates these responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals, antibodies, and reagents
Male Sprague Dawley rats (250–270 g) were purchased from Charles River Laboratory (Wilmington, MA). The Institutional Animal Care and Use Committee of Rhode Island Hospital/Brown University approved all the protocols. Rabbit polyclonal antiphosphorylated cAMP response element binding (pCREB) (Ser133) antibody was obtained from Upstate (catalog no. 06-519; Temecula, CA). Rabbit antiproTRH83–106 (pEH24), antiproTRH115–151 (pAV37), antipreproTRH207–255 (pYE17), and anti-TRH antiserum were developed in our laboratory (17). Dr. Seidah (IRCM, Montréal, Canada) donated rabbit anti-PC1/3 and anti-PC2 antibodies. Fluorescent secondary antibodies and streptavidin-Alexa fluor 594 were from Molecular Probes (Eugene, OR). Alkaline phosphatase-linked goat antirabbit antibody was from Bio-Rad Laboratories (Richmond, CA). Biotinylated goat antirabbit antibody was from Jackson ImmunoResearch Laboratories (West Grove, PA). All the reagents were purchased from Sigma-Aldrich (St. Louis, MO), except when indicated.

Hypothalamic cultures
Hypothalamic neurons were cultured as previously described (18). In brief, diencephalic tissue was dissociated by neutral protease digestion. Cells were cultured in L15-DMEM containing 10% fetal calf serum and supplemented with various additives, including 50 mM 5-bromo-2'-deoxyuridine during the first 4 d, as previously described (18). After 12 d in culture, hypothalamic neurons (5 x 106/flask) were stimulated with increasing concentrations of NE (0–100 nM) for a period of 1–3 h in serum-deprived medium. After incubation, media were collected, evaporated using a speed vacuum system, reconstituted in RIA buffer, and TRH measured by RIA methods. Cells were collected in either TRIzol Reagent (Invitrogen Inc., Carlsbad, CA) for RNA isolation or extraction buffer [50 mM Tris/HCl (pH 7.2), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS) freshly supplemented with an inhibitor cocktail] for analysis of PCs. Samples per time and NE concentration were run at least in triplicate. All experiments were run in triplicate.

Quantification of preproTRH, PC1/3, and PC2 mRNAs by Northern blot
The mRNA analysis was performed as previously described (14). Briefly, samples were homogenized in Trizol (Invitrogen) according to the manufacturer’s specifications; RNA concentration and quality was determined by spectrophotometry and ethidium bromide-stained formaldehyde/agarose gel electrophoresis, respectively. Equal amounts of total RNA were separated in an 1.0% agarose/formaldehyde gel, blotted onto a nylon filter, and hybridized according to published methods (19). Filters were washed and then exposed to a PhosphorImager screen (Fujix BAS 1000, Tokyo, Japan) for digitized data collection and analysis. Filters were then stripped and rehybridized with different probes. The hybridization probes were rat preproTRH, PC1/3, PC2, and mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA labeled with {alpha}-32P-dCTP using a random-priming kit (Roche Diagnostics Corp., Indianapolis, IN).

Quantification of PC1/3 and PC2 protein by Western blot analysis
Western blot analysis was performed as previously described (20). Briefly, samples were homogenized, centrifuged, supernatants removed, and subjected to protein determination using the Bradford assay (Pierce, Rockford, IL). An equal amount of total protein in each well was applied onto 8% glycine-SDS-PAGE gels. Precision Plus protein standards (Bio-Rad Laboratories) were used as molecular mass markers. After electrophoresis, proteins were electroblotted onto polyvinyl difluoride membranes (Millipore Laboratories, Billerica, MA) and blocked. Membranes were probed with a 1:1000 dilution of antibodies against PC1/3 (87 kDa) and PC2 (75 and 68 kDa) as described earlier (20). The immunoreactive bands were visualized using an alkaline phosphatase-linked goat antirabbit antibody followed by Immunostar assay as described by the manufacturer (Bio-Rad Laboratories).

Radiolabeling experiments
The radiolabeling experiments were performed as previously described (21). Hypothalamic neurons were stimulated with NE (0–100 nM) for 6 h in low leucine, serum-deprived medium. For proTRH and processing product labeling, cells were pulsed for 6 h with 0.3 mCi of [3,4,5-3H] leucine; for TRH labeling, 0.3 mCi of [2,3,4,5-3H] proline was used. After the incubations, medium was removed for TRH analysis; cells were collected in 2 N acetic acid for proTRH processing analysis. For TRH analysis, medium was immunoprecipitated with anti-TRH antibodies, as described previously (21). Immunoprecipitated were fractionated on an isoelectric focusing gel electrophoresis as previously described (21). Briefly, samples were run in 30% acrylamide/1% bisacrylamide gel tubes in buffer containing 6 M urea and 1% [(3-cholamidopropyl) dimethyl-ammonio]-1-propane-sulfonate. Pharmalyte 2.5–5 and Pharmalyte 5–8 were added to the solution to generate a pH 4.6–8.0 gradient. After the run, each gel tube was sliced, put in scintillation vials, and counted. The pH gradient was determined by using proteins with known isoelectric points as standards (21). For proTRH and processing product analysis, cell extracts were boiled, sonicated, and centrifuged. Supernatants were immunoprecipitated with anti-proTRH115–151 (pAV37) antibody as described before (21). This antiserum recognizes 26, 16.5, 15, 10, and 4.5 kDa moieties (Fig. 1Go). Immunoprecipitates were fractionated onto a tricine/SDS-PAGE system (22). After electrophoresis, gels were sliced (Hoefer Scientific Instruments, San Francisco, CA), put in scintillation vials, and counted. Peptide molecular masses were identified using commercial molecular mass markers (Diversified Biotech, Newton, MA). This experiment was repeated on three independent occasions.


Figure 1
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  		FIG. 1. Diagrammatic representation of the rat proTRH prohormone (light gray) and part of its processing to TRH and non-TRH peptides. The TRH progenitor sequence region is indicated by black rectangles. The sites at which PC1/3 and PC2 produce their enzymatic cleavage is indicated by arrows. Peptides recognized by antipreproTRH83–106 (pEH24) antibodies are depicted in dark gray, and the peptides recognized by anti-preproTRH115–151 (pAV37) antibody are depicted in white.

 
In vivo studies
To study the effect of acute cold exposure, rats were divided in two groups: control rats, maintained at controlled room temperature (22 C), and cold-exposed rats, transferred to an environmental chamber at 4 C. To evaluate the role of different adrenoreceptor antagonists, rats were stereotaxically implanted with an intracerebroventricular (ICV) guide cannula (Plastics One, Roanoke, VA) as previously described (16). The placement coordinates for the lateral ventricle were: antero-posterior, –0.8 mm, lateral, –1.2 mm, and ventral, –3.6 mm. After surgery, animals were allowed to recover for at least 7 d. The correct placement of the cannulas was verified by the angiotensin test (16). On the day of the experiment, animals were given with 4 µl ICV of vehicle [artificial cerebrospinal fluid (aCSF)], propanolol (0.3 µg/rat), or phentolamine (3 µg/rat) dissolved in aCSF. After 5 min, subsets of rats from each group were either maintained at 22 C or transferred in an environmental chamber at 4 C. Initially, we tested several doses of the antagonists, and we chose a dose for each one that did not affect the basal activity of the HPT axis (data not shown). Stress increases levels of hypothalamic pCREB; hence, to evaluate the role of this protein on the response to cold exposure, rats were rapidly and stress-free anesthetized via a jugular catheter implanted 2 d before the experiment (16).

Animals were killed by decapitation after being either 1 or 3 h under the controlled temperature environment. Blood was collected for TSH, and T4 analysis. The PVN and ME were rapidly removed from the hypothalamus by surgical dissection, as previously described (16). For RNA isolation, PVN samples were collected in TRIzol reagent (Invitrogen) from rats exposed during 1 h to the experimental paradigm because the preproTRH mRNA increase is rapid and transitory (11). PVN and ME samples from rats exposed during 3 h were collected in either 2 N acetic acid freshly supplemented with a protease inhibitor cocktail for peptide extraction or extraction buffer for PC analysis. Each group contained five to 10 animals per treatment per time point. For immunohistochemistry studies, rats were perfused 45 min (for pCREB and proTRH colocalization) or 3 h (for PC and proTRH colocalization) after cold exposure as previously described (16). Brains were frozen and cut in 25-µm-thick coronal sections on a sliding cryostat. We used three rats per group per time point.

For RT-PCR analysis, all samples were done in parallel as previously described (14). Briefly, samples were homogenized, and RNA concentration and quality of each isolate was determined as described above. Primer sequences used for PCR are depicted in Table 1Go. We used 17 cycles to amplify GAPDH cDNA and 25 cycles for the PCR quantification (14). Each PCR was incubated with 1.0 µl of {alpha}-32P-dCTP (29.6 TBq/mmol, 370 MBq/ml) (PerkinElmer, Torrance, CA). Amplified samples were subjected to acrylamide gel electrophoresis; gels were then transferred to filter paper, dried, and subjected to 32P quantification by PhosphorImager analysis. For PC analysis, PVN samples were homogenized, and processed for Western blot as described above. For peptide extraction, PVN samples were sonicated in acetic acid as previously described (20); supernatants were collected, and protein concentration was determined by Bradford assay (Pierce). Supernatants were used to measure TRH by RIA methods, or further fractionated in a SDS-polyacrylamide gel to measure proTRH peptides. In this case, gels were cut into 2-mm slices in a gel slicer (Hoeffer Scientific Instruments) after electrophoresis. Peptides were extracted from gel slices in acetic acid. These fractions were then evaporated and reconstituted in RIA buffer.


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  		TABLE 1. Primers sequence used for the PCR

 
Double immunohistochemistry
Double pCREB/proTRH staining was performed as described (16). Sections were pretreated with 1% H2O2, 0.3% glycine, and 0.03% SDS. Sections were then blocked, and incubated with anti-pCREB antibody (1:1500) overnight at 4 C. The next day, sections were treated with biotinylated goat antirabbit antibody (1:1000) and then with Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) according to the manufacturer’s protocols. Visible signal resulted in a brown precipitate. Consecutively, sections were incubated overnight with antiproTRH antibody (antipreproTRH207–255; 1:5000). The next day, sections were washed and incubated with a fluorescent goat antirabbit Alexa 594 (red) antibody. Sections were then mounted on glass slides and coverslipped with Vectashield mounting media containing 4',6'-diamino-2-phenylindole (Vector Laboratories). Because both antibodies are from rabbit, controls staining were run to confirm that each antibody by itself stains with the same pattern. Results were visualized using either fluorescence (proTRH) or bright-field light (pCREB) sources. Fluorescent images (12 bit) and diaminobenzidine (DAB) images (24 bit) were acquired with an E800 microscope (Nikon Inc., Melville, NY) and a Spot II digital camera (Diagnostic Instruments, Sterling Heights, MI). All analyses were carried out under the same optical and light conditions. Using Adobe Photoshop (Adobe Systems Inc., San Jose, CA), fluorescence and bright-field photographs were combined. The percentage of proTRH neurons containing labeled nuclei was determined at medial and posterior level of the PVN, in which most of the hypophyiotropic proTRH neurons are located (23). ProTRH neurons with visible 4',6'-diamino-2-phenylindole-positive nuclei and proTRH neurons with visible pCREB-positive nuclei on each side of the third ventricle were counted. The relationship was expressed as a percentage, which represents pCREB-positive proTRH neurons, compared with the total number of proTRH neurons observed. Additionally, pCREB-staining intensity inside each proTRH-positive cell was quantified using National Institutes of Health (NIH) image software (Springfield, VA). The intensity measurement is the mean gray of the pixels inside the nuclear perimeter and is expressed as arbitrary units of intensity (AUI).

Colocalization with anti-PC and anti-proTRH antibodies was performed using the tyramide signal amplification (TSA) method (PerkinElmer, Boston, MA) that allows simultaneous localization using two antibodies generated in the same animal species. Double localization was carried out according to the manufacturer’s instructions. First, sections were incubated with antiproTRH (antipreproTRH207–255; 1:200,000) antibody and then visualized with the TSA method using streptavidin-Alexa 594. Then sections were incubated with either anti-PC1/3 or anti-PC2 antibody (1:1000), which was detected with goat antirabbit Alexa 488 antibody (14). Sections were mounted on glass slides and coverslipped with Vectashield mounting media (Vector Laboratories). Confocal images were acquired with a Nikon PCM 2000 using argon (488) and green helium-neon (543) lasers. Adobe Photoshop was used to convert the images to RGB and for assembly of the figures (Adobe Systems). Colocalization of PC1/3 or PC2 with proTRH was expressed in percentages, which represent either PC1/3- or PC2-positive proTRH neurons, compared with the total number of proTRH neurons observed in the medial and posterior level of the PVN.

RIA analyses
All RIAs used in this study were developed in our laboratory and already described (5). Tracers were iodinated using the chloramine T method followed by HPLC purification. RIA to measure TRH was performed using the TRH antiserum that recognizes only mature TRH peptide. For the detection of proTRH-derived peptides, we used antiproTRH83–106 (pEH24) antiserum, which recognizes 4.8-, 3.8-, and 2.8-kDa moieties (see Fig. 1Go) (20). All RIAs were performed in duplicate; the sensitivity of the TRH and pEH24 assays were 2.0 and 40.0 pg/tube, respectively. The intra- and interassay variability were 5–6 and 9–12%. Plasma T4 level was determined using MP Biomedicals commercial kit (Diagnostic Division; Orangeburg, NY). TSH level was determined using a RIA developed by A. F. Parlow (National Hormone and Pituitary Program, Harbor-UCLA Medical Center, Torrance, CA) (14). The sensitivity of TSH and T4 assays were 0.5 ng/ml and 1.2 µg/dl, respectively. The intra- and interassay variability were approximately 5–7 and 10–11%, respectively.

Statistical analyses
Data are expressed as the mean ± SEM. Data were analyzed by either Student’s t test or ANOVA followed by Newman Keuls posttest for comparison of different mean values. Significant differences were considered when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
NE increases preproTRH mRNA levels, proTRH peptides, and TRH secretion in cultures of hypothalamic neurons
Initially, we treated neurons with increasing concentrations of NE (0–100 nM) for 3 h followed by Northern blot analysis. The NE-induced increase of preproTRH mRNA peaked after 1 h of treatment (data not shown) and remained elevated within the studied period. Figure 2AGo shows that NE treatment increased preproTRH mRNA expression in dose-response fashion after 3 h of treatment. NE treatment (100 nM) increased preproTRH mRNA from 77.9 ± 7.3 to 155.5 ± 10.0 AUI (P < 0.05). We also studied NE action on the biosynthesis of proTRH protein using a radiolabeling strategy. Figure 2BGo shows that NE treatment dose-dependently increased all the proTRH moieties recognized by antiproTRH115–151 antibody. Three independent experiments showed that NE (100 nM) caused approximately 3-fold increase in proTRH precursor and other proTRH-derived peptides. NE also increased approximately 3-fold the release of de novo synthesized TRH (21) (Fig. 2CGo). As shown in Fig. 2DGo, NE acted as secretagogue independent of the protein biosynthesis because it increased TRH release after 1 h of incubation, when newly formed TRH is still not available for secretion (24). TRH values, according to RIA analyses, were 3.9 ± 0.3, 5.5 ± 0.1, and 6.8 ± 0.2 pg/ml for NE treatment 0, 10, and 100 nM, respectively.


Figure 2
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  		FIG. 2. NE increases preproTRH mRNA levels, proTRH biosynthesis, and TRH release in a dose-response manner in primary hypothalamic cultures. A, Northern blot analysis for preproTRH and GAPDH mRNAs. The figure shows a phospho image of preproTRH (1.7 kb) and GAPDH (1.4 kb) mRNA bands. The lower panel shows integrated OD for each band from three independent experiments using NIH Image software. B, Radiolabeling of NE-treated hypothalamic neurons with 3H-leucine for 3 h as compared with untreated cells. Extracted peptides were immunoprecipitated with anti-pAV37 antibody, separated in a SDS-polyacrylamide gel, followed by gel slicing and counting. C, NE-treated hypothalamic neurons radiolabeled with 3H-proline for 3 h. Labeled peptides in the media were immunoprecipitated with anti-TRH antibody and resolved on an isoelectric focusing gel electrophoresis, followed by gel slicing and counting. D, Measured released TRH by RIA after 1 h of NE treatment. Values are mean ± SEM. *, P < 0.05 vs. NE at 0 nM.

 
NE increases PC1/3 and PC2 mRNA and protein levels in primary cultures of hypothalamic neurons
Because NE stimulates proTRH biosynthesis, we wanted to determine whether NE also up-regulated PC1/3 and PC2. Figure 3AGo shows that NE-induced increase of PC1/3 and PC2 mRNA expression was transient, being highest after 1 h of treatment. In contrast to preproTRH mRNA, PC mRNA levels returned to basal levels after 3 h of treatment. NE treatment (100 nM) for 1 h increased PC1/3 mRNA from 220 ± 15 to 344 ± 22 AUI (P < 0.05, Fig. 3BGo) and PC2 mRNA from 258 ± 16 to 352 ± 30 AUI (P < 0.05, Fig. 3CGo). Western blot analysis demonstrated that NE treatment resulted in a dose-response increase of PC1/3 and PC2 protein levels after 3 h of treatment. NE treatment (100 nM) increased PC1/3 protein from 259 ± 21 to 508 ± 42 AUI (P < 0.05, Fig. 3DGo) and PC2 protein from 342 ± 33 to 512 ± 39 AUI (P < 0.05, Fig. 3EGo).


Figure 3
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  		FIG. 3. NE increases PC mRNA levels and protein biosynthesis in primary hypothalamic cultures. A, Northern blot analysis for PC1/3 and PC2 and GAPDH mRNAs in hypothalamic neurons treated with 100 nM of NE. B and C, Integrated OD for PC1/3, and PC2 bands using NIH Image software. The data are a representation of three independent experiments. D and E, Western blot analysis for PC1/3 and PC2 from hypothalamic neurons treated for 3 h with increasing concentrations of NE. Molecular mass for PC1/3 and PC2 is indicated at the right side. The lower panels show a comparison of the integrated OD for PC1/3 and PC2 using NIH Image software. Values are mean ± SEM. *, P < 0.05 vs. NE at 0 nM.

 
Cold exposure in rats mimics the NE-induced changes seen in cultures of hypothalamic neurons
Some studies implicate NE as the main neurotransmitter mediator of the HPT axis activation in response to cold exposure (12). Therefore, we determined whether the PVN neurons of rats exposed to cold respond in a similar manner to the NE-induced changes on TRH neurons in vitro. Cold exposure showed to activate the HPT axis outputs and increase TRH and PC biosynthesis in the PVN (Table 2Go). We found that preproTRH, PC1/3, and PC2 mRNA levels increased in approximately 1.5-, approximately 2-, and approximately 1.5-fold in the PVN (P < 0.05) at 1 h of cold exposure. After 3 h in a cold environment, TRH peptide and total pEH24-related peptide levels (the algebraic sum of all femtomoles in each peptide form measured by pEH24 RIA after gel electrophoresis) were also increased in the PVN, compared with control rats. TRH peptide levels significantly decreased in the ME, presumably because of neurosecretion of this peptide because plasma TSH and T4 hormones were significantly increased at the same time point. Results depicted in Table 2Go also show that the PVN of cold-exposed rats had an increase of approximately 100% and approximately 50% in PC1/3 and PC2 protein levels, respectively.


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  		TABLE 2. Cold exposure activates the HPT axis and the PCs in the hypothalamic PVN of rats

 
Because the results described above indicate that cold exposure increases both proTRH and PCs in the PVN, we wanted to test whether this occurs in TRH neurons by doing colocalization studies of proTRH and PC1/3 or proTRH and PC2 in brain slices. Figure 4AGo depicts a representative image showing the colocalization of PCs and proTRH in the PVN from rats exposed to 4 C or room temperature. PCs showed different localizations within cells; whereas PC1/3 staining was mostly concentrated in the perinuclear area, PC2 staining was more homogenously distributed within the cell body. Double staining in the PVN of control rats showed partial colocalization between PC1/3 and proTRH (Fig. 4AGo, control upper panels) or between PC2 and proTRH (Fig. 4AGo, control lower panels). The percentage of proTRH neurons positive for PC1/3 and PC2 were 46.5 ± 5.5 and 19.0 ± 8.9, respectively. In cold-exposed rats, there was an increase in the intensity of the fluorescent signal for each protein (data not shown). We also observed a significant increase in the amount of double staining between PC1/3 and proTRH (Fig. 4BGo, cold upper panels) and between proTRH and PC2 (Fig. 4CGo, cold lower panels). In cold-exposed rats, the percentage of proTRH neurons positive for PC1/3 and PC2 were 91.2 ± 7.9 and 75.2 ± 8.1, respectively (P < 0.05).


Figure 4
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  		FIG. 4. Cold exposure for 3 h increases PC1/3 and PC2 protein levels specifically in TRH neurons in the hypothalamic PVN nucleus. A, Series of confocal images from rat PVN, which are immunoreactive to proTRH, PC1/3, and PC2. The left and right vertical panels show colocalization images in low and high magnification (see arrows). For proTRH detection (red) we used the commercial TSA method, and for the PCs, we used a direct fluorescent antirabbit antibody (green). Orange staining denotes colocalization between proTRH and PCs. B and C, The bar graphs represent the statistical analysis comparison in percentages of TRH neurons positive for either PC1/3 or PC2. Values are mean ± SEM. *, P < 0.05 vs. control. Magnification, x200 (left) and x600 (right). Scale bar, 50 µm (left) and 20 µm (right).

 
Cold exposure activates the biosynthesis and proteolytic processing of proTRH in the hypothalamic PVN via ß-adrenoreceptors
After determining that cold exposure mimics in vitro NE effects in proTRH neurons, we decided to corroborate the specific role of NE on this activation by using a pharmacological approach. To block {alpha}-adrenoreceptors, we used phentolamine, a nonselective {alpha}-antagonist, and to block ß-adrenoreceptors, we used propanolol. Figure 5Go shows the effect of both treatments on the cold-induced activation of the HPT axis (after 3 h). Importantly, basal activity of the HPT axis was not affected by these treatments. At the peripheral level, both {alpha}- and ß-adrenoreceptor antagonists blocked the cold-induced activation of the HPT axis, as determined by the reduction of the cold-induced elevation of plasma TSH and T4 (Fig. 5Go, A and B). At the hypothalamic level, adrenoreceptor antagonists had different effects. Propanolol pretreatment significantly blocked both cold-induced increase in TRH peptide in the PVN and cold-induced decrease of TRH in the ME (Fig. 5Go, C and D). Also, propanolol-pretreated rats exposed for 1 h to cold did not have a cold-induced increase of preproTRH mRNA in the PVN (1.01 ± 0.05, normalized by GAPDH mRNAs, and expressed as fold stimulation). In contrast, whereas phentolamine pretreatment blocked the cold-induced decrease of TRH in the ME, the cold-induced increase in TRH in the PVN remained similar to the control group (Fig. 5Go, C and D). Moreover, phentolamine-pretreated rats exposed for 1 h to cold environment had an increase of preproTRH mRNA in the PVN (1.31 ± 0.06, normalized by GAPDH mRNAs, and expressed as fold stimulation).


Figure 5
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  		FIG. 5. Cold-induced activation of the HPT axis is dependent on both {alpha}- and ß-adrenergic receptors. The panels shown in this figure represent comparative values of specific RIAs for plasma TSH (A), plasma T4 (B), TRH in PVN (C), and TRH in the ME (D). For this experiment eight animals were used in each condition. Rats were ICV pretreated with a ß-adrenoreceptor antagonist [propanolol (Prop) 0.3 µg/rat], {alpha}-adrenoreceptor antagonists [phentolamine (Phent), 3 µg/rat], or vehicle. Five minutes later rats were exposed to cold environment (4 C) or room temperature (22 C) during 3 h followed by decapitation. Values are the mean ± SEM. ANOVA was followed by a multiple comparison using a Newman-Keuls test. Values are mean ± SEM. *, P < 0.05 vs. control.

 
We next determined whether ß-adrenoreceptor signaling also regulates PC levels. Western blot results showed that cold-exposed rats had an increase of approximately 70% in PC1/3 levels in the PVN (Fig. 6AGo). Propanolol pretreatment abolished this cold-induced increase, whereas phentolamine pretreatment of rats did not affect the cold-induced increase of PC1/3. Analysis of PC2 (68 kDa) showed similar results as seen for PC1/3: cold exposure induced an approximately 50% stimulation in PC2 level, which was fully blocked by propanolol and unchanged by phentolamine pretreatment (Fig. 6BGo). To evaluate whether these changes in PC levels affect proTRH processing, we examined the SDS-PAGE separation profile of proTRH-derived peptides from the PVN. We used an antiserum raised against pEH24 (preproTRH83–106), which recognizes several intermediate forms (25) (Fig. 1Go). None of these treatments affected the basal biosynthesis of proTRH as judged by the SDS-PAGE separation followed by RIA for pEH24 (data not shown). Figure 6CGo shows that the total amount of pEH24-related peptides (the algebraic sum of all femtomoles in each peptide form) increased in the PVN of cold-exposed rats, compared with control rats, from 19.9 ± 0.4 to 33.9 ± 3.3 fmol/µg of protein (P < 0.05). Propanolol pretreatment reversed the cold-induced increase of pEH24-related peptides (17.8 ± 1.9 fmol/µg of protein). However, phentolamine pretreated rats had high pEH24-related peptide levels in the PVN (34.6 ± 5.3 fmol/µg of protein, P < 0.05 vs. control group). Analyses of the percentage of the different proTRH-related peptides recognized by anti-pEH24 antibody revealed that its processing in the PVN was altered by cold exposure (Table 3Go). Whereas approximately 35% and approximately 18% of the total femtomole amount of peptides of control rats belonged to the pro-pEH24 and TRH-pEH24 forms, respectively, only approximately 28% and approximately 11% were measured under the cold condition (P < 0.05 for both forms). In contrast, pEH24 peptide in the PVN of cold-exposed rats increased approximately 1.3-fold over control rats (~48% vs. ~61%). Table 3Go shows that propanolol reversed the cold-induced increase in processing of proTRH, whereas {alpha}-adrenoreceptor antagonist did not alter it.


Figure 6
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  		FIG. 6. Cold-induced increase in posttranslational processing of proTRH, and PCs are dependent on ß-adrenergic receptors. A and B, Typical Western blots for PC1/3 (A) and PC2 (B) from rat PVN samples in animals subjected to 4 C or room temperature (22 C) during 3 h. The upper panels depict a typical Western blot (two animals per group), whereas the lower panels show the average integrated OD of three different Western blots (mean ± SEM). NIH image software was used to obtain integrated ODs. The molecular masses for PC1/3 and PC2 are indicated in upper panels. ANOVA was followed by a multiple comparison using a Newman-Keuls test. *, P < 0.05 vs. control. C, Representative analysis of an electrophoretic separation of PVN samples extracted from rats that were ICV pretreated with a ß-adrenoreceptor antagonist [propanolol (Prop) 0.3 µg/rat], {alpha}-adrenoreceptor antagonists [phentolamine (Phent), 3 µg/rat], or vehicle and then exposed to cold environment (4 C) during 3 h. Control rats were kept at room temperature (22 C). Separation was performed in tricine SDS-PAGE followed by gel slicing, acid extraction of gel slices, and RIA for pEH24 peptide. Molecular masses of the identified peaks are based on the migration pattern of molecular mass standards. Graph represents a typical profile of three independent experiments.

 

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  		TABLE 3. Cold exposure for 3 h alters the ratio for each proTRH-derived peptide in the PVN via ß-adrenoreceptors

 
Cold-induced activation of TRH neurons in the hypothalamic PVN correlates with activation of pCREB signaling
Finally, we determined the specific intracellular signaling that mediates the cold-induced activation of TRH neurons in the PVN. To test this, we performed a costaining approach using antiproTRH and anti-pCREB antibodies. Figure 7Go depicts a set of representative images from each experimental group. Brown nuclear staining represents pCREB, and red cytoplasmic staining represents proTRH staining. Low- and high-magnification merged images depicted in Fig. 7AGo (left and right vertical panels) show the colocalization of pCREB and proTRH. Control rats treated with aCSF showed some pCREB staining in the PVN mainly in the magnocellular subdivision, consistent with previous reports (23, 26). A massive increase of neurons with nuclear pCREB staining was seen 45 min after cold exposure, particularly in the parvocellular PVN (Fig. 7Go, arrows). Quantitative analysis indicated that 20.6 ± 2.9% of parvocellular TRH cells were positive for pCREB in the medial and posterior parts of the PVN in aCSF-treated/room temperature rats; conversely, pCREB labeling in TRH neurons significantly increased to 42.3 ± 4.8% in cold-exposed rats (P < 0.05, Fig. 7BGo). In addition, cold exposure increased DAB staining intensity in TRH neurons from 36.8 ± 1.5 to 47.7 ± 2.4 AUI (P < 0.05, Fig. 7CGo). Phentolamine pretreatment failed to affect the cold-induced increase of pCREB staining (Fig. 7Go, B and C). However, propanolol pretreatment fully blocked the cold-induced increase of pCREB staining (Fig. 7Go, B and C).


Figure 7
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  		FIG. 7. The activation of TRH neurons after 45 min of cold exposure is accompanied with an increase in pCREB signaling in the PVN. A, Series of confocal images from rat PVN, which are immunoreactive to proTRH and pCREB. The left and right vertical panels show colocalization images in low and high magnification (see arrows). Double immunohistochemistry was performed using a direct fluorescent antirabbit antibody against proTRH antibodies (red), and a peroxidase coupled antirabbit antibody followed by DAB precipitation to detect pCREB antibodies (brown). B and C, Bar graphs representing the statistical analysis of the images. B, Percentage of proTRH neurons positive for pCREB for each treatment. C, The intensity of the nuclear DAB staining within the TRH neurons of each experimental group. Quantifications analysis was done using NIH image software. Prop, Propanolol; Phent, phentolamine. Values are mean ± SEM. *, P < 0.05 vs. control. Magnification, x100 (left) and x400 (right). Scale bar, 100 µm (left) and 20 µm (right).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we demonstrated that NE is a potent stimulator of proTRH biosynthesis and the main mediator of the cold-induced activation of the HPT axis at the hypothalamic level. This activation occurs via ß-adrenoreceptors, which mediate both the synthesis and proteolytic processing of proTRH in the PVN, presumably via pCREB signaling. In vitro, we found that NE affects several steps in the biology of TRH, such as gene transcription, protein biosynthesis, and secretion; these changes were also coupled with an up-regulation of PCs.

Previous studies demonstrated that cold exposure increases preproTRH mRNA in the PVN, TRH secretion from the ME, and plasma levels of TSH and T4 (9, 10, 11). The preproTRH mRNA rapidly and transiently increases in the PVN under cold exposure; it peaks in the 1–2 h of exposure (10, 11) and remains elevated for at least 48 h if cold persists (9). There is some evidence that NE afferent systems mediate the effect of cold on the HPT axis (12, 13). This activation might occur at two levels because NE terminals make synaptic contact with TRH neurons in the PVN and TRH fibers in the ME (13, 27). Our results demonstrate that both levels are important in the cold-induced activation of the TRH neurons. The {alpha}-antagonist phentolamine fully blocked the cold-induced activation of the HPT axis at the plasma level, likely resulting from an inhibition of TRH secretion from the ME because TRH content did not decrease in this area (Fig. 5Go). Agreeing with our data, Tapia-Arancibia et al. (28) found that activation of {alpha}1-adrenoreceptors in the ME induces TRH release. Central phentolamine pretreatment did not abolish the cold-induced up-regulation of proTRH biosynthesis and processing in the hypothalamic PVN of rats. These effects were instead mediated by ß-adrenoreceptors because they were fully blocked by propanolol pretreatment. To our knowledge, this is the first evidence that {alpha}- and ß-adrenoreceptors play a different role in the regulation of TRH neurons, in which ß-adrenoreceptor signaling is the main regulator of the cold-induced increase of TRH biosynthesis.

Our data also support the concept that phosphorylation of CREB is an initial step in the cold-induced activation of the parvocellular TRH neurons. ß-Adrenoreceptors are well-known activators of pCREB signaling (29), and it has been shown that NE induces cAMP accumulation in the hypothalamus via ß-adrenoreceptors (30). Then the increase of pCREB in TRH neurons that can be completely blocked by propanolol, and the presence of ß-adrenoreceptors in the PVN (31), supports a role for this pathway in the cold-induced activation of TRH neurons. The pCREB transcription factor strongly up-regulates preproTRH mRNA expression via its binding to a critical sequence in the preproTRH promoter called site 4 (32, 33). The pCREB signaling also activates PC promoters: CREB response elements have been reported in PC1/3 and PC2 promoters, and they are up-regulated by activating the cAMP pathway (34, 35, 36, 37). Therefore, NE via pCREB signaling might directly and simultaneously activate the synthesis of both preproTRH and PC mRNAs in the hypophysiotropic TRH neurons.

The basal activity of the HPT axis was also down-regulated when higher doses of adrenoreceptor antagonists were tested (not shown). This observation is consistent with the relatively high basal levels of TRH neurons positive for pCREB (around 20%) in control animals and suggests that some adrenergic inputs are acting even in a basal condition. However, if pCREB activation persists whereas cold stimulus continues, it can explain a conflicting observation: How can cold-induced activation of TRH cells persist in the presence of a concomitant rise of circulating T3, which is a potent inhibitor of these neurons (9)? Because both pCREB protein and thyroid receptor bind to the same sequence in the preproTRH promoter (site 4), and we have shown that they have opposite action on the TRH promoter (38), it is likely that the presence of high cold-induced pCREB levels overrides the negative feedback mediated by T3. Interestingly, PC1/3 and PC2 promoters also contain negative thyroid response elements, and T3 negatively regulates PC1/3 and PC2 levels in the PVN of rats (15, 39, 40, 41). Then it is possible that pCREB signaling on the PC promoters in the PVN also overrides the T3-induced decrease of PCs. Future studies will be needed to corroborate this possibility. In basal conditions, we found that about half of the TRH neurons contain PC1/3 immunoreactivity, similar to others studies (42, 43). This is surprising because of the critical role of this enzyme in the processing of proTRH (5, 22). We speculate that all TRH neurons in the PVN express PC1/3; however, basal levels of PCs might be undetectable by immunohistochemical method in some of these neurons. When TRH neurons are activated (like in cold exposure), PC1/3 expression is up-regulated and enzyme levels become detectable in most of the TRH neurons, supporting the importance of this enzyme in proper and efficient proTRH processing.

NE up-regulates proTRH processing via an increase of the PC-processing enzymes. One could hypothesize that other steps in the biosynthesis of TRH could be regulated. In studies done with the Cpefat/fat mice, we demonstrated that the deficiency of CPE affects TRH biosynthesis (7). However, CPE levels remain unaltered in the PVN of cold-exposed rats (not shown), suggesting that this enzyme is not an important checkpoint at which the processing of proTRH is regulated. Conversely, PAM is believed to be a rate-limiting enzyme because its levels can be regulated under physiological conditions (44). Recently we found that PAM activity is responsive to chronic changes in plasma thyroid levels. Using the ratio TRH to TRH-Gly as a measure of PAM activity, we found that it increases in the PVN of rats subjected to low iodine/propylthiouracil diet-induced hypothyroidism, and conversely, PAM activity decreases in T4-treated rats (1). However, the ratio TRH to TRH-Gly was not altered in the PVN in cold-exposed rats, even when both TRH and TRH-Gly levels increased (not shown). These data suggest that PAM activity is not acutely regulated by cold exposure. Therefore, although other steps in the biosynthesis of TRH could potentially be regulated, our data indicate that PC enzymes exert the major control of proTRH processing.

Specific regulation of prohormone processing is likely another key step during which final amounts of bioactive peptides can be tightly regulated. Different factors, such as thyroid hormones, leptin, the melanocortin system, and NE, potently stimulate proTRH biosynthesis in concert with an increase in the PC enzymes in the hypothalamic PVN (14, 15, 16). In another example, we recently showed that regulation of POMC biosynthesis also occurs at the posttranslational level through coordinated changes in the PC enzymes in hypothalamus and medulla (45). In addition, pro-CRH or provasopressin prohormones are coregulated with the PCs (46). Therefore, we propose that this could be a common mechanism used by different types of cells to generate bioactive peptides in a more efficient way. Thus, the coordinated regulation of PCs may play an important role in neuroendocrine cells in maintaining a proper enzyme-substrate homeostasis and ensuring adequate processing of newly synthesized prohormones. In summary, this study provides novel information on the regulation of TRH biosynthesis in hypophysiotropic PVN neurons and identifies potential drug targets in pathological states associated with central HPT axis dysfunction, such as hypo- or hyperthyroidism conditions, or obesity.


   Acknowledgments
 
We thank Virginia Hovanesian for her assistance in the acquisition of the images. Also, we thank Bill Tsiaras for reading this manuscript.


   Footnotes
 
This work was supported by the Dr. George A. Bray Research Scholars Award Fund (to M.P.) and National Institute of Diabetes and Digestive and Kidney Diseases/National Institutes of Health (NIH) Grants R01 DK58148 and R01 National Institute of Neurological Disorders and Stroke/NIH Grant NS045231 (to E.A.N.).

Author Disclosure Statement: The authors have nothing to declare.

First Published Online June 21, 2007

Abbreviations: aCSF, Artificial cerebrospinal fluid; AUI, arbitrary units of intensity; CP, carboxypeptidase; DAB, diaminobenzidine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPT, hypothalamic-pituitary-thyroid; ICV, intracerebroventricular; ME, median eminence; NE, norepinephrine; PAM, peptidylglycine {alpha}-amidating monooxygenase enzyme; PC, prohormone convertase; pCREB, phosphorylated cAMP response element binding; PVN, paraventricular nucleus; SDS, sodium dodecyl sulfate; TRH-Gly, TRH progenitor, Gln-His-Pro-Gly.

Received April 23, 2007.

Accepted for publication June 13, 2007.


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 Introduction
 Materials and Methods
 Results
 Discussion
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