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Endocrinology Vol. 142, No. 2 896-906
Copyright © 2001 by The Endocrine Society

ARTICLES

PreproTRH178–199 and Two Novel Peptides (pFQ7 and pSE14) Derived from Its Processing, Which Are Produced in the Paraventricular Nucleus of the Rat Hypothalamus, Are Regulated during Suckling1

Eduardo A. Nillni, Fraser Aird, Nabil G. Seidah, Roberta B. Todd and James I. Koenig

Division of Endocrinology (E.A.N., R.B.T.), Department of Medicine, Brown University School of Medicine, Rhode Island Hospital, Providence, Rhode Island 02903; Maryland Psychiatric Research Center (J.I.K.), University of Maryland School of Medicine, Baltimore, Maryland 21228; Department of Psychiatry and Behavioral Sciences (F.A.), Northwestern University Medical School, Chicago, Illinois 60611; and Laboratory of Biochemical Neuroendocrinology (N.G.S.), Clinical Research Institute of Montréal, Montréal, Québec H2W1R7, Canada

Address all correspondence and requests for reprints to: Dr. Eduardo A. Nillni, Division of Endocrinology, Rhode Island Hospital, 55 Claverick Street, Room 400/430, Providence, Rhode Island 02903. E-mail: Eduardo_Nillni{at}Brown.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Suckling increases preproTRH messenger RNA in hypothalamic paraventricular neurons (PVN) and also markedly increases TRH release during the first period of lactation. Whether lactation alters preproTRH processing resulting in the generation of novel proTRH-derived peptides that may be involved in the regulation of PRL secretion lactation is not known. Therefore, in the present study we determine whether some other peptides derived from proTRH potentially contribute to lactation-induced PRL secretion. We have recently demonstrated that two members of the family of prohormone convertases PC1 and PC2 play a significant role in proTRH processing. PC1 is the major contributor in proTRH processing, whereas PC2 may have a specific role in cleaving TRH from its extended forms. In this study, we used a recombinant vaccinia virus system to coexpress rat preproTRH complementary DNA with PC1, PC2, and the neuropeptide 7B2 in GH4C1 cells (somatomammothophs, rat). We found that two novel peptides, preproTRH178–184 (pFQ7), and preproTRH186–199 (pSE14), were formed after the cleavage of their precursor preproTRH178–199 (pFE22) by only PC2. Their formation was confirmed by microsequence analysis. Anatomical analyses revealed that these peptides are also found in the rat PVN. In addition, we found that pFE22, pSE14 and pFQ7 produced a dose-dependent release of PRL from primary cultures of pituitary cells compared with one of the well studied secretagogues of PRL, TRH. To establish whether these peptides might play a role in vivo in the regulation of PRL release, we took rat litters on postnatal day 4, separated the pups from their mothers for 6 h, and then reunited the pups and mothers for 45 min. At the end of this period, the mothers were killed, acidic extracts of microdissected PVN were prepared and subjected to SDS-PAGE, followed by slicing and analysis by pFE22 RIA. Forty-five minutes of suckling induced a marked 6-fold increase in serum levels of PRL. In addition, PVN levels of pFE22 and pSE14 increased approximately 5-fold during the same period in the acutely suckling females. Lactating animals that were separated from their litters and never reunited with their pups had low levels of PRL, and pFE22 and pSE14. These data provide the first evidence for alterations in proTRH processing in the PVN during lactation and suggest that the products of this altered processing may play a physiological role in the regulation of PRL secretion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
RAT preproTRH is a 29-kDa polypeptide composed of 255 amino acids. This precursor contains an N-terminal 25 amino acid leader sequence followed by an N-terminal flanking peptide, five copies of the TRH progenitor sequence Gln-His-Pro-Gly flanked by paired basic amino acids (Lys-Arg or Arg-Arg), four non-TRH peptides lying between the TRH progenitors, and a C-terminal flanking peptide (1). The N-terminal flanking peptide (preproTRH25–50-R-R- preproTRH53–74) is further cleaved at the C-terminal side of the arginine pair site to render preproTRH25–50 and preproTRH53–74, thus yielding a total of seven proTRH-derived peptides.

The most abundant preproTRH-derived peptide is the tripeptide, TRH, which is synthesized in the PVN and at other sites in the brain (1, 2). TRH is responsible for the biosynthesis and secretion of TSH from the anterior pituitary (3, 4). TSH, in turn, stimulates thyroid hormone biosynthesis and release (5, 6). TRH also influences the release of other hormones, including PRL, GH, vasopressin and insulin (7, 8, 9), and the classic neurotransmitters noradrenaline and adrenaline (10). In comparison to the known roles of TRH, there is little information about the biological activities of the other proTRH-derived peptides. The most studied proTRH-derived peptide, preproTRH160–169 (also known as Ps4 or TRH-potentiating peptide) enhances TRH-stimulated TSH release from the anterior pituitary and stimulates TSHß gene promoter activity (11). This peptide also potentiates TRH-induced-gastric acid secretion when microinjected into the dorsal motor nucleus of the vagus (12). Recently, considerable attention has focused on another peptide derived from this precursor, notably preproTRH178–199, which is also produced in the PVN. This molecule (also known as pFE22) is reported to be a corticotropin-inhibiting factor, which inhibits ACTH release, and through reductions of POMC messenger RNA (mRNA) also inhibits its synthesis (13, 14, 15). pFE22 also appears to have significant anxiolytic activity (14). However, little else is known about the production and biological effects of peptides derived from the preproTRH precursor. Thus, clues to the roles of proTRH-derived peptides other than TRH must come from an examination of their regional distribution, or evidence of regulation under specific physiological or pathological conditions.

In the present study, using an antibody against the pFE22 sequence, we further characterized the posttranslational processing of the C-terminal peptides derived from preproTRH. Furthermore, we report for the first time that some of these novel peptides derived from preproTRH may have a unique biologic role, and that the production of pFE22 and preproTRH186–199 (pSE14) may be regulated physiologically during the suckling period.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
All normal and timed-pregnant female Sprague Dawley rats used in these studies were purchased from Charles River Laboratories, Inc. (Wilmington, MA, and Kingston, NY). The experimental protocols and euthanasia procedures were reviewed and approved by the Institutional Animal Care and Use Committees of Rhode Island Hospital/Brown University or the University of Maryland School of Medicine.

Tissue culture
Mouse corticotropic AtT-20 cell line. AtT-20 cells transfected with a complementary DNA (cDNA) encoding preproTRH were grown in 75 cm2 flasks at 37 C in an atmosphere of 5% CO2, 95% air, and 90% humidity. Each flask was plated with 2 x 106 cells and cultures were maintained for 7 days in a DMEM (Life Technologies, Inc., Gaithersburg, MD) containing 10% FCS as previously described (16). Culture medium was replaced every 2 days with fresh medium. Experiments were performed in 75 cm2 flasks containing between 25–30 x 106 cells with a total protein content of 10.5 ± 0.3 mg (n = 6). The protein content (Bio-Rad Protein Assay (Bio-Rad Laboratories, Inc. Richmond, CA) of each flask was determined to correct for small differences in cell number between flasks.

Primary cultures of hypothalamic neurons. Hypothalamic neuronal cultures were produced as previously described (17). In brief, timed pregnant female rats on day 17 of gestation were anesthetized with pentobarbital (60 mg/kg), the abdominal cavity was opened, and the fetuses were removed. Each fetus was decapitated, and the diencephalon was isolated. Diencephalic tissue was dissociated to single cells by neutral protease digestion (1 U/tissue, Sigma, St. Louis, MO). The cells were cultured for up to 14 days in L-15-DMEM (D-MEM/liter-15) (18) containing 10% FCS (Life Technologies, Inc.) and supplemented with various additives (17). Before plating, all wells were coated with poly-D-lysine (20 µg/ml, Sigma). For immunocytochemistry (ICC), the cells were plated on four-chamber glass LabTek (Nunc Inc., Naperville, IL) slides (106 cells/ml). For radiolabeling experiments, the cells were incubated in 25 cm2 flasks (5–6 x 106 cells/flask).

Primary cultures of pituitary cells. Anterior pituitary (AP) cells from female rats (Sprague Dawley) were cultured as previously described (19). Briefly, AP tissue was separated from posterior/intermediate lobes, collected into sterile HBSS, enzymatically dispersed with neutral protease (1.5 U/AP) (Sigma) and plated in a monolayer on 24-well plates at a density of 2 x 105 cells/well. The cells were cultured with in a modified L-15/DMEM (Life Technologies, Inc.) supplemented with 10% steroid-free FCS, streptomycin and penicillin (19). After 48 h in culture, the cells were washed and preincubated for 18 h with the same media containing 1% FCS. At the end of 18 h, the spent media was removed and fresh media containing test substances was added (n = 6). After 30 min of incubations with the appropriate treatment, release media and cell content was harvested for RIA analysis of pituitary hormones as standard in our laboratory.

Infection of GH4C1 cells by vaccinia virus recombinants
The coinfections of GH4C1 cells (50 x 106 cells) with either a recombinant vaccinia virus containing the sequences for PC1 or PC2, or combination thereof, (VV:mPC1, VV:mPC2, and VV:m7B2) and preproTRH were performed at one plaque forming unit per cell as previously reported (20, 21, 22, 23). Following infection, cells were washed and resuspended in serum free media for 4 h, as previously reported (20, 22, 24). Cell media were collected, lyophilized, washed, resuspended, and analyzed using SDS-PAGE and RIA. Cellular peptide content was not measured because preliminary results indicated that most of the peptides were contained in the media fraction.

Radiolabeling experiments
AtT-20 cells. Experiments were conducted in 75 cm2 flask containing approximately30 x 106 cells. Before radiolabeling, cells were incubated for 30 min with 6 ml of labeling media (9 volumes of leucine-free DMEM mixed with 1 volume of regular media) containing 2.5% dialyzed FCS. Then cells were pulsed with 300 µCi of (3,4,5, 3H)-Leucine (156 Ci/mmol) for 4 h before harvesting. Following incubation, the media were removed and radiolabeled peptides were extracted as previously described (25).

Hypothalmic neurons. On day 12 in culture, each flask of hypothalamic neurons containing 5 x 106 cells per flask was pulsed with 300 µCi of (3,4,5, 3H)-leucine (156 Ci/mmol) in leucine-free DMEM containing 3% FCS for 20, 60, and 120 min. After the incubations, the medium was removed and the cells washed three times with HBSS containing 0.1 mg/ml of cold leucine. After the last wash the cells were rapidly cooled on ice, and 2 ml of 2 N acetic acid containing 2 mM EDTA, 2 mM EGTA and enzyme inhibitors (phenylmethylsulphonylfluoride, aprotinin, bacitracin, bestatin, and pepstatin, each at 0.1%) were added. The cells were scraped and heated to 95 C for 10 min before sonication. One hundred microliters of sample was removed for protein assay. The remainder of the cell extract was centrifuged at 15,000 rpm for 30 min. The supernatant was then lyophilized and held at -20 C until electrophoresis on SDS-PAGE.

Immunoprecipitation
An immunoprecipitation protocol was carried out as described previously (25). Briefly, lyophilized cell extracts were resuspended in 10 µl of 0.2% BSA and 200 µl of hypotonic buffer A (10 mM NaPO4, pH 7.2/1 mM EDTA/0.1% Triton X-100). Following resuspension, cell extracts were incubated for 24 h at 4 C with 1:500 dilution of protein G purified anti-proTRH178–199 (26). Then, 1:1000 dilution of goat-antirabbit IgG was added along with 75 µl of buffer B (500 mM KCl/50 mM NaH2PO4, pH 7.4/5 mM NaEDTA/0.25% Triton X-100). Samples were further incubated for 4 h at 4 C. Immunoprecipitates of cell extracts were washed once with buffer B and once with buffer C (10 mM NaH2PO4, pH 7.2/15 mM NaCl), which removes EDTA and Triton X-100. The immunoprecipitates were then resuspended in sample buffer (0.0625 M Tris, pH 6.8/1% SDS/15% glycerol/15 mM dithiothreitol) and boiled for 4 min before SDS-PAGE. Immunoprecipitation using nonimmune serum and immune serum directed against proTRH178–199 in the presence of an excess of synthetic proTRH178–199 peptide did not result in visible peaks (not shown).

SDS-PAGE
Radioactive or cold samples were fractionated by loading them onto a discontinuous Tricine-PAGE (SDS-PAGE) system for separation of low molecular weight peptides (20). Following electrophoresis, gels were cut into 1 mm slices in a gel slicer (Hoefer Scientific Instruments, San Francisco, CA) and prepared for either counting or RIA. For tritium analysis, immunoprecipitated peptides were extracted from gel slices by incubation in 1 ml of 1 N acetic acid for 24 h at 4 C. Scintillation fluid (Bio Safe II, RPI, IL) was added and samples were counted in a scintillation counter. Preparation for RIA included the same acetic acid extraction as described above, but, following incubation, gel slices were removed. Samples were then lyophilized and resuspended in the appropriate RIA buffer. Recovery of peptides from gel slices has been shown to be approximately 90% as determined by RIA before and following the electrophoresis. To identify the apparent molecular weight of fractionated peptides on SDS-PAGE, a series of molecular weight markers were used. Prestained BSA, 80.0 kDa; ovalbumin, 49.5 kDa; carbonic anhydrase, 32.5 kDa; soybean trypsin inhibitor, 27.5 kDa; lysozyme, 18.5 kDa (Bio-Rad Laboratories, Inc.); trypsin inhibitor, 20.4 kDa; myoglobin, 16.95 kDa; myoglobin fragment IV, 14.4 kDa; myoglobin fragment III, 8.16 kDa; myoglobin fragment II, 6.2 kDa; myoglobin fragment I, 2.5 kDa (Diversified Biotech, Newton, MA).

Synthetic peptides
PreproTRH178–184 (pFE14) and preproTRH186–199 (pSE7) were synthesized in the Quality Control Biochemical facilities (Quality Control Biochemicals, Hopkington, MA) from the deduced amino acid sequence of the preproTRH peptide (corresponding to amino acids Phe-Ile-Asp-Pro-Glu-Leu-Gln and Ser-Trp-Glu-Glu-Lys-Glu-Gly-Glu-Gly-Val-Leu-Met-Pro-Glu of the precursor).

In vitro processing and micro sequencing analysis
Purified mouse PC2 preparation (kindly donated by Dr. Iris Lindberg from Louisiana Medical Center, New Orleans, LA) was preincubated for 1 h at 37 C in a buffer containing 100 mM CaCl2, 1 M sodium acetate pH 5.6 and Brij 35 (1%). This mixture was then incubated with pFE22, digested for 0, 6 and 24 h at 37 C, hydrolyzed, and sequenced. Fitting of the amino acids was compared with the known sequence of the peptide preproTRH178–199 (FINPELQRSWEEKEGEGVLMPE) and its fragments. Micro sequencing analysis was performed as previously described (27).

Peptide RIAs
pFE22 RIA. The dried gel slices were dissolved in 100 µl buffer C (63 mM Na2HPO4, 13 mM EDTA, 3 mM NaH3, 0.1% Triton X-100, 250 kallikrein inhibitor units (KIU)/ml Aprotinin (Sigma). Twenty-five microliters of each sample were used to measure pFE22 immunoreactivity by RIA. To each sample was added 75 µl of buffer C, and 100 µl of a 1:4,000 dilution (in buffer A; 63 mM Na2HPO4, 13 mM EDTA, 3 mM NaH3) of a rabbit antiserum raised against rat pFE22 (this antibody was generated in rabbit against the whole preproTRH178–199 sequence). No cross-reactivity of anti-pFE22 antiserum was observed with any of the proTRH-derived peptide assays currently used in our laboratory. The samples were mixed and incubated at 4 C for 48 h. One hundred microliters of 125I-labeled pFE22 tracer were added, and the samples were mixed and incubated at 4 C for 24 h. The tracer, in which the Phe at amino acid position 178 was replaced by Tyr, was iodinated by the chloramine-T method and diluted to 10,000 cpm/100 µl in buffer C. To each sample, 500 µl of a second antibody solution (buffer A plus 0.3 mg/ml goat antirabbit immunoglobulin [P3; Antibodies Incorporated, Davis, CA] was added. This was followed by 1:500 dilution of normal rabbit serum [Life Technologies, Inc., Grand Island, NY], 4% [wt/vol] polyethylene glycol. The samples were vortexed and incubated at 23 C for 45 min. After centrifugation at 1,600 x g for 30 min at 4 C, the supernatants were decanted and the pellets counted in a {gamma} counter.

RIA using reagents and protocols obtained from the National Hormone and Pituitary Program (Bethesda, MD) measured PRL and TSH levels in media and serum. All RIAs were performed on the same volume of material, in triplicate. The inter and intraassay coefficients of variation for the preproTRH178–199 assay are 7 and 4%. The inter and intraassay coefficients of variation for the PRL assay are 6 and 2%.

Double staining immunocytochemistry (dICC)
Hypothalamic neurons (3 x 105) from 12-day-old cultures were fixed with 4% paraformaldehyde in PBS and subjected to an immunocytochemistry protocol as we previously described (17, 20). Immunoreaction with primary antibody was performed at 4 C for 24 h. Goat antirabbit immunoglobulin conjugated with fluorescein isothiocyanate was used as the fluorescence marker. A wide range of dilutions for the primary (anti-pFE22) and secondary antibodies were tested. The optimal dilutions were found to be 1:1,000 for the primary antibody and 1:2,000 for the secondary with an incubation time of 24 h at 4 C for the primary antibody and 2 h at room temperature for the secondary antibody. Control experiments including the incubation of cells without primary antibody or preimmune sera, and the blocking of the primary antibody with the synthetic pFE22 peptide for which the antibody was generated, were performed and did not show any positive staining. The microtubule associated protein 2 (MAP2) monoclonal antibody was detected with Texas red conjugated to sheep antimouse IgG.

In vivo studies
Timed-pregnant female Sprague Dawley rats were purchased from Charles River Laboratories, Inc. (Kingston, NY). The animals arrived on day 5 of pregnancy. All animals were maintained in a temperature and humidity controlled facility with ad libitum water and food. Animals were maintained on 12-h light, 12-h dark cycle (lights on 0700 to 1900 h). Pups were delivered vaginally following 22 days of gestation. On postnatal day 4, the pups were separated from their mothers for 6 h. Pups were placed in another room in a plastic animal cage on a heating blanket at 37 C to maintain their body temperature. After 6 h, randomly selected litters were returned to their mothers. After 45 min of suckling, the mothers were separated from their pups and killed by decapitation. Lactating mothers not reexposed to their litters were killed after 6 h and 45 min of separation from their litters. All animals were killed between 1500 h and 1600 h by rapid decapitation. Trunk blood was collected in plastic tubes and allowed to clot. Serum was separated by low speed centrifugation, and following transfer to another tube, was stored frozen until PRL analysis. Brains were removed from the skull and the median eminence (ME) was dissected from the fresh brain under microscopic control as previously described (28). The isolated ME was placed in a microfuge tube containing 200 µl 2 N acetic acid with enzyme inhibitors, and boiled for 15 min to extract proTRH-derived peptides before freezing on dry ice. The reminder of the brain was frozen on powdered dry ice for later dissection of the PVN. A 1-mm section was taken from the frozen brain immediately caudal to the optic chiasm. This section is known to contain the PVN (28). The frozen tissue was placed in 200 µl of 2 N acetic acid with enzyme inhibitors and boiled for 15. The samples following boiling were placed in dry ice. The extracted PVN samples and ME were then subjected to SDS-PAGE followed by slicing and RIA as described above.

Statistics
RIA values were plotted against the gel slice number corresponding to a particular molecular mass peptide generated graphs. Protein assay results were used to correct for minor variations in total cell number. Data were displayed as ng/ml. ANOVA followed by a multiple comparison (Tukey-Kramer test) was employed when appropriate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of proTRH-derived peptides and characterization of anti-pFE22 antibodies
To characterize the proTRH-derived peptides products the anti-pFE22 antibody recognizes, we evaluated 3H-leucine radiolabeled peptides immunoprecipitated from primary cultures of hypothalamic neurons, which contain an endogenous source of proTRH, and in a tumor cell line (AtT-20) expressing transfected preproTRH cDNA. In vivo samples from extracted adult rat PVN and ME tissues were also analyzed. The PVN contains proTRH and proTRH-derived peptides, and the ME, which contains only fibers and nerve terminals is expected to contain mostly fully processed peptides rather than precursor molecules of proTRH.

Figure 1Go, A and B depicts a typical electrophoretic separation pattern of immunoprecipitated 3H-labeled peptides extracted from transfected AtT-20 cells and hypothalamic neurons after 2 h of labeling. Three distinct moieties of molecular mass (MM) about 10, 5.6, and 2.6 kDa were observed in AtT20 cells. Hypothalamic neurons showed a similar peptide profile to the AtT-20 cells with the addition of new smaller form of MM about 1.7 kDa. The 2.6-kDa peptide had the same mobility as an iodinated pFE22 standard. Figure 1CGo shows a typical electrophoretic pattern of extracted peptides from female rat PVN detected by the anti-pFE22 RIA. Similar to AtT-20 cells and hypothalamic neurons, the PVN showed the presence of the 10-, 5.6-, 2.6-kDa moieties plus a smaller form of about 1.7 kDa. In all these tissues, with the exception of ME, the most prominent preproTRH-derived peptide observed was the 10-kDa form, which we proposed in earlier studies (25) to be preproTRH160–255. In addition, pulse labeling experiments done over time in AtT-20 cells revealed that after 30 min of labeling only the 10 kDa peptide was detected. After a 60 min pulse, the 5.6- and 2.6-kDa peptides were visible (data not shown), which increased at 120 min as shown in Fig. 1AGo. A second peptide of about 5.6 kDa was also prominent in experiments shown in panels A–C of Fig. 1Go. This peptide could represent preproTRH160–199, as suggested in our early publication (25), which contains the pST10-TRH-pFE22 sequence. Interestingly, transfected AtT-20 cells apparently did not produce the 1.7-kDa peptide. RIA analysis of electrophoresed peptides derived from the ME revealed, as expected, the presence of only final products of preproTRH processing. Figure 1DGo shows two immunoreactive peptides of about 2.6 and 1.7 kDa MM. To further characterize the specificity of the antibody, we immunoprecipitated 3H-leucine peptides obtained from hypothalamic cultures with anti-pFE22 followed by further incubations with 100 nM of cold pSE14 and pFQ7 peptides. As depicted in Fig. 2Go both peptides were able to displace the radioactive peaks corresponding to the peptides recognized by these antibodies. However, the strongest inhibition was with pFQ7. This indicates that both epitopes are recognized by this antibody.



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  		Figure 1. Electrophoretic separation of immunoprecipitated 3H-peptides with anti-pFE22 and extracted peptides from brain samples followed by RIA. The immunoprecipitates or extracted peptides from PVN and ME were electrophoresed on an SDS-polyacrylamide gel. The counts from each slice were plotted against gel slice, and the peptides from fractionated brain samples were subjected to specific RIA against pFE22. Molecular masses of the identified peaks are indicated based on the migration of standards. These figures represent a typical profile of three independent experiments. The iodinated pFE22 run in parallel with the samples is indicated in the graph. Panels A and B show the profile of 3H-peptides recognized by anti-pFE22 antibodies in AtT-20 cells transfected with preproTRH and in primary cultures of hypothalamic neurons. Panels B and C show the profile of RIA-peptides recognized by anti-pFE22 antibodies in samples extracted from PVN and ME. ED 80 represents the dose of peptide that causes 20% reduction in binding from B0 or maximum binding.

 


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  		Figure 2. Electrophoretic separation of immunoprecipitated 3H-peptides with anti-pFE22 and in the presence of cold pSE14 and pFQ7. The immunoprecipitates of extracted peptides from radiolabeled primary cultures of hypothalamic neurons were electrophoresed on a SDS-polyacrylamide gel. The counts from each slice were plotted against gel slice. To displace the radioactive binding between anti-pFE22 and radiolalabeled peptides produced de novo in hypothalamic neurons, 50 µg/ml of cold pSE14 and pFQ7 were added to the mixture and incubated overnight followed by double immunoprecipitation as described in Materials and Methods. Molecular masses of the identified peaks are indicated based on the migration of standards. These figures represent a typical profile of three independent experiments.

 
Immunolocation of proTRH-derived peptides in primary cultures of hypothalamic neurons using anti-pFE22
Analysis of the subcellular distribution of these peptides indicated that this antibody specifically labels the axonal filaments of hypothalamic neurons (Fig. 3AGo). This suggests that the intermediate forms and end products of processing detected by this antibody are formed in secretory granules (SG) of the regulated secretory pathway (2). The red staining for the MAP2 marker showed, as expected, staining in the dendrites, but no staining for preproTRH-derived peptides was observed in this compartment (Fig. 3BGo).



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  		Figure 3. Immunocytochemical staining of primary cultures of hypothalamic neurons using anti-pFE22. Neuronal cells cultured for up to 12 days in four chamber LabTek slides were fixed with 4% paraformaldehyde followed by immunoreaction with anti-pFE22. Fluorescein isothiocyanate conjugated to goat antirabbit globulin was used as a probe (green color). The marker MAP enriched dendrites are indicated with red color (Texas red). Panel A shows axons single stained with anti-pFE22 using goat antirabbit IgG FITC conjugated. Panel B shows double labeling of axon with anti- pFE22 (green) and MAP (red).

 
Role of PC1 and PC2 in the formation of preproTRH178–199 and two novel peptides
We next determined the role of PC1 and PC2 in the generation of the 2.6-kDa peptide (preproTRH178–199). In previous studies, using the vaccinia virus system for coinfection of preproTRH with members of the prohormone convertase family (20), we found that PC1 was the primary enzyme involved in the processing of preproTRH and that PC2 played a role later in that process. For example, PC2 was more active in the formation of TRH by processing the TRH-pEH24 extended peptide (20). In this set of experiments, with the availability of pFE22 antiserum, we determined the fate of the processing products, i.e. pST10 and pFE22, derived from the 10-kDa precursor peptide by the action of PC1 and PC2.

Using the recombinant vaccinia virus system, we coexpressed rat preproTRH cDNA with the prohormone convertases PC1, PC2, and neuropeptide 7B2 cDNAs in the endocrine GH4C1 cell line. RIAs analysis of the secreted products revealed that PC1 was more effective in cleaving preproTRH to immunoreactive forms recognized by anti-pFE22 serum, whereas PC2 seems to play a minor role, even in the presence of 7B2 (Fig. 4Go). 7B2 (29) is a peptide shown to be important for the maturation and regulation of proPC2 activity (27, 29), and thus is a necessary component in confirming the cleavage capability of PC2. However, when we analyzed the same samples after fractionation by gel electrophoresis followed by RIA analysis, we found that the cleavage specificity for PC2 differed from that for PC1. For example, in cells coinfected with PC1 and preproTRH, two prominent moieties of about 5.6 kDa and 2.6 kDa were formed (Fig. 5AGo). The latter had the same mobility as synthetic pFE22. On the other hand, when the cells were coinfected with PC2 and preproTRH, the 5.6-kDa peptide was not detected; instead, the 2.6-kDa peptide and a smaller form of about 1.7 kDa were observed (Fig. 5BGo). A triple infection of preproTRH, PC1 and PC2 cDNAs is depicted in Fig. 5CGo, which shows the presence of the 5.6-, 2.6-, and 1.7-kDa peptides.



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  		Figure 4. Immunoreactivity to pPE22 in GH4C1 cells coinfected with preproTRH, PC1, PC2, and 7B2 cDNAs. RIAs were performed against resuspended serum-free media. Cell means of recognized products in nanograms are plotted against the indicated coinfected construct. The data represents a mean value of six identical wells per condition from three independent experiments, with P < 0.05 on Tukey-Kramer.

 


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  		Figure 5. Electrophoretic separation of media from coinfected GH4C1 cells with proTRH, PC1, and PC2. Nanograms of peptide obtained by RIA in each extraction are plotted against gel slice. Molecular masses of the identified peaks are indicated based on the migration of standards. The iodinated pFE22 run in parallel with the samples is indicated in the graph.

 
In vitro processing of pFE22 by PC2
Because PC2 has different cleavage specificity than PC1, we sought to identify the region of the pFE22 sequence where PC2 produces its endoprotelytic cleavage(s). Therefore, we incubated purified PC2 with purified pFE22 peptide, and the products were subjected to microsequence analysis. The pFE22 sequence contains an Arg residue at preproTRH185 and a Lys residue at preproTRH190. Results from microsequence analysis revealed that only one specific cleavage at Arg185 (... Glu182-Leu183-Gln184-Arg185-{downarrow}-Ser186-Trp187-Glu188-Glu189-Lys190 ... ) was produced (Fig. 6Go). This enzymatic reaction generated two peptides that were heretofore undescribed, notably pFQ7 and pSE14 (0.84 and 1.7 kDa, respectively). The 1.7-kDa peptide has the same apparent molecular mass as the peptide detected in the adult rat PVN and ME, and in primary cultures of hypothalamic neurons (Fig. 1Go, B–D), strongly suggesting that these peptides are processing products of pFE22 in vivo.



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  		Figure 6. Amino acid sequence analysis of cleaved pFE22 with purified PC2. The in vitro processing of pFE22 peptide was performed by incubations with PC2 at 37 C for 6–24 h. After purification on RP-HPLC on a Vydac C18 column, the eluted samples were then hydrolyzed for 24 h in 6 N HCl and amino acid analysis was performed on a Beckman Coulter, Inc. (Fullerton, CA). 3600 amino acid analyser. Fitting of the amino acids was compared with the known sequence of the pFE22 peptide and its fragments. Only a major cleavage was observed at the basic Arg residue, preproTRH185 generating a fragment of 7 and 14 amino acids. ND represents UV reading but not amino acids presence.

 
Biologic role of pFE22, pSE14 and pFQ7 peptides in pituitary cells
In an effort to determine whether these peptides have any effects on pituitary hormone secretion, we synthesized pFQ7 and pSE14 peptides and analyzed their potential biologic activity in primary cultures of rat pituitary cells along with their precursor form, pFE22. To this purpose, we evaluated the effect of pFE22 and its processing products as compared with TRH, a well-established PRL secretagogue, in a dose-response manner. The effect on TSH release in a dose- response manner was also measured. To conduct these experiments, we used well established incubation conditions for monitoring PRL release by TRH (30) and compare that effect with pFE22, pFQ7 and pSE14. As shown in Fig. 7Go, TRH stimulated PRL release in a dose-dependent manner reaching a plateau at approximately10-8 M. Whereas, the proTRH-derived peptides pFE22, pFQ7 and pSE14 needed 10-7 M concentration to reach a plateau. In addition, TRH showed to stimulate higher levels of PRL release than the non-TRH peptides. Because these peptides are not known to be TSH releasing hormone or to have similar structure to TRH, one would not expect to have a significant TSH releasing activity on the pituitary cells. The data presented in Fig. 7Go confirms that hypothesis.



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  		Figure 7. Stimulation of PRL release by pFE22, pFQ7, pSE14 and TRH inanterior pituitary cells. Cultured AP cells were incubated with various concentrations of TRH, pFE22, pFQ7, and pSE14 at 37 C for 30 min, and then the release media was assayed for PRL or TSH RIAs. The data represent a mean value of six identical wells per condition from three independent experiments, with P < 0.05 on Tukey-Kramer.

 
Regulation of pFE22 and pSE14 peptides levels in the PVN during suckling
Suckling was shown to increase both preproTRH mRNA in PVN and ME TRH release during the first period of lactation (31). In our experiments (Fig. 7Go) pFE22 and its products were found to be PRL secretagogues in primary pituitary cultures. Therefore, we analyzed the processing and quantitative changes of proTRH-derived peptides in the PVN during suckling in vivo using anti-pFE22 antibodies. Twelve lactating female rats separated from their pups for 6.75 h had serum PRL levels of approximately 5 ng/ml (Fig. 8AGo). Reexposure of other six previously separated, lactating mother rats to their litters for 45 min following a 6 h separation induced a 12-fold increase in serum PRL concentrations (P < 0.001). Analysis of brain preproTRH processing was performed in the PVNs and MEs of the separated and lactating female rats (group of six rats per condition) used to generate the serum PRL data as in Fig. 8AGo. Figure 8Go, B and C, shows typical gel electrophoresis patterns of preproTRH processing as detected with a pFE22 RIA in a 6 h-separated, nonsuckled female rat and a female rat exposed to her litter for 45 min after separation. Figure 8BGo shows that during suckling, the 5.6-, 2.6-, and 1.7-kDa peptides were substantially increased as compared with nonsuckling controls. These later two peptides represent pFE22 and pSE14, respectively. At least a 5-fold increase in PVN pFE22 and pSE14 amounts was observed in the suckled female rats. In the nonsuckling controls, large peaks of about 16.5 kDa and 10 kDa were observed in the PVN. The 16.5-kDa moiety could represent the precursor peptide to the 10-kDa peptide previously described (16) and other smaller forms described in this study. Interestingly, a similar larger peak was observed in the PVN of normal female rats (Fig. 1CGo). However, the smaller peptides (pFE22 and pSE14) were present in the PVN of the separated, nonsuckled female rats in only minute amounts, suggesting that suckling induced the processing of the larger preproTRH peptides to smaller preproTRH fragments that have notable PRL-releasing activity. Similarly to the PVN, in the ME an increase in the accumulation of peptides of about 2.6 (pFE22) and 1.7 kDa (preproTRH186–255) MM peptides was observed (Fig. 8CGo). However, this accumulation in the ME of suckled rats did not shown to be much higher than those from nonsuckled rats.



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  		Figure 8. Regulation of pFE22 and pSE14 peptide levels in the PVN and ME during Suckling. Panel A depicts the level of PRL in serum from 12 animals in a 6 h separated, nonsuckled female rats (six) and female rats (six) exposed to their litter for 45 min after separation. Panel B and C show an electrophoretic pattern of preproTRH processing products from the PVN and ME as detected with a pFE22 RIA in 6 h separated, nonsuckled female rats and female rats exposed to their litter for 45 min after separation.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Posttranslational processing of prohormone proteins, usually at pairs of basic residues, is crucial for maturation of bioactive peptides contained within a given precursor (2, 32, 33). During the last few years, we have provided unequivocal evidence for the significant role of PC1 in preproTRH processing and have shown that PC2 may have a specific role in cleaving smaller forms of the processed precursor (17, 20, 34, 35).

In this study, we have further characterized the processing of the intervening preproTRH178–199 peptide closer to the C-terminal side of the preproTRH molecule, identified two novel peptides, and proposed a potential physiologic role for some of these moieties during lactation. The identification of the processing products was accomplished using an antibody against the pFE22 sequence, which was used to either detect or purify C-terminal preproTRH fragments in the PVN, ME, primary cultures of hypothalamic neurons and in transfected AtT-20 cells expressing preproTRH cDNA. The role of PC1 and PC2 in the formation of the novel peptides was determined using a recombinant vaccinia virus system to coexpress rat preproTRH cDNA with the prohormone convertases PC1, PC2 and neuropeptide 7B2 in GH4C1 cells.

Electrophoretic separation of radiolabeled peptides from cultured cells or cold peptides extracted from brain tissue revealed that this antibody recognizes four moieties of about 10, 5.6, 2.6, and 1.7 kDa, consistent with our previous proposed model of preproTRH processing. The 1.7-kDa (proposed pSE14) form described for the first time in this study was not present in transfected AtT-20 cells. This supports the hypothesis that pFE22 is cleaved only by PC2 because those cells have very low endogenous levels of PC2 and further supports our coinfection and in vitro processing studies. The anatomical distribution of these peptides were almost exclusively in the secretory granules of axonal processes. This suggests that after the initial cleavage of the prohormone, which is produced in the trans-Golgi network (16, 36), these peptides are probably produced in the secretory granules. Evidence for this hypothesis was previously proposed in trafficking studies conducted in AtT-20 cells encoding preproTRH cDNA (16, 36).

Our coinfection results indicated that PC1 is responsible for the cleavage of the 10-kDa and 5.6-kDa peptides to produce pFE22, whereas PC2 has only a minor role in the generation of these peptides, even in the presence of 7B2, a chaperone neuropeptide essential for the maturation of PC2 (29). However, analysis of the fractionated samples from coinfections with preproTRH and PC1, preproTRH and PC2 and in vitro processing plus sequencing analysis revealed that PC2 specifically cleaves at the monobasic residue Arg185 to generate two novel peptides, pFQ7 and pSE14, which were formed by the cleavage of pFE22.

As was shown for many regulatory peptide precursors, processing of these molecules can occur at single and/or pairs of basic residues. There is a pair of basic residues of either Lys-Arg or Arg-Arg between each TRH molecule and its connecting sequences, which was shown to be cleaved primarily by PC1 (20). Here we show for the first time that PC2 can cleave at the preproTRH186 monobasic residue. By comparing amino acids around the monobasic cleavage sites, it was previously suggested that these cleavages follow certain sequence motifs, and they can be described by the rules that govern monobasic cleavages (37). These basic rules are: 1) A basic amino acid is present at either 3, 5, or 7 amino acids N-terminal to the cleavage site; 2) Hydrophobic aliphatic amino acids (leucine, isoleucine, valine, or methionine) are never present in the position C-terminal to the monobasic amino acid at the cleavage site; 3) A cysteine is never present in the vicinity of the cleavage site; and 4). An aromatic amino acid is never present at the position N-terminal to the monobasic amino acid at the cleavage site. In addition to these rules, the monobasic cleavages follow certain tendencies: 1) The amino acid at the cleavage site is predominantly arginine; 2) The amino acid at the position C-terminal to the cleavage site tends to be serine, alanine, or glycine in more than 60% of the cases; 3) The amino acid at either 3, 5, or 7 position N-terminal to the cleavage site tends to be arginine; 4). Aromatic amino acids are rare at the position C-terminal to the monobasic amino acid at the cleavage site; and 5) Aliphatic amino acids tend to be in the two positions N-terminal to and the two positions C-terminal to the cleavage site, except as noted above. The cleavage reported here by PC2 fits all these rules with the exception of rule one in the first set of rules. Figure 9Go shows a diagrammatic representation of rat preproTRH and its cleavage by PC1 and PC2 as proposed from our previous and present studies (20, 34, 35).



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  		Figure 9. Proposed model of processing for the C-terminal side of the TRH prohormone. This cartoon represents an extension of the original processing model for proTRH (1 ). The additional pathway of processing added to this model was determined using the new anti-pFE22 antibody. The small arrows indicate the sites where PC1 and PC2 produce their enzymatic cleavages. Big arrows indicate the direction of processing. The inverted Y represents the anti-pFE22 antibody situated where the pFE22 sequence is located within the proTRH molecule.

 
In our current model of preproTRH processing, we propose that after the first two alternative cleavages of preproTRH by PC1 (2, 20), two set of peptides can be generated. The C-terminal 16.5-kDa and N-terminal peptide 9.5-kDa peptides, and the N-terminal 15-kDa and C-terminal 10-kDa peptides (2, 20, 25). Therefore, the 10-kDa peptide seen in these studies could originate by PC1 action on either of the two alternative proteolytic cleavage products, following the first cleavage of the prohormone at the pair of basic residues 107–108 or 113–114 to generate the 16.5-kDa intermediate peptide or after the first cleavage at the pair of basic residues 152–153 or 158–159 to generate the 10-kDa peptide. We propose that the 10-kDa peptide is further processed at the pair of basic residues 200–201 or 206–207 by PC1 to generate the 5.6 kDa (pST10-TRH-pFE22) peptide (25). This peptide is then processed to produce pFE22, which is later cleave by PC2 to generate pFQ7 and pSE14 peptides (Fig. 9Go).

The search for physiological PRL-releasing factors has proceeded since TRH, GnRH, and somatostatin were first isolated and characterized in the 1960s and 70s. A number of peptides, including TRH and VIP, have been postulated to fill this role (38, 39, 40, 41, 42, 43). Along with recent studies suggesting that novel factors derived from the posterior pituitary could also be involved (44, 45). In the present studies, we report for the first time that pFE22 and two novel small peptides, pFQ7 and pSE14, that are derived from the processing of the pFE22 molecule, all exhibit PRL-releasing activity when incubated with dispersed anterior pituitary cells in vitro. Furthermore a physiological role for these peptides in regulating PRL secretion is suggested by our demonstration that PVN and ME tissue extracts taken from acutely suckling female rats contain enhanced levels of pFE22 and pSE14. In initial studies done in our laboratory, we found that the pFE22 peptide binds specifically to GH3 cell membrane as a first indication for the presence of a receptor for this peptide. Interestingly, in nonsuckled animals an accumulation of a peptide similar in size to the 16.5-kDa form was observed. This may suggest that the 10-kDa peptide is the precursor to the 5.6-kDa form, whereas pFE22, pSE14 and pFQ7 may derive from the 16.5-kDa intermediate peptide. The peptides detected in our in vivo samples arise from the proteolytic action of PC1 and PC2 on the preproTRH molecule present in PVN neurons, as shown in our previous, and present studies by mRNA coexpression, protein colocalization, and biochemical processing (2, 17, 20). These peptides are also found in the ME, which could reflect their release into the hypophysial portal vasculature. The median eminence did not showed to have a significant difference between the two conditions, even though was higher in the suckled animals, in peptide content for pFE22 and pSE14 compared with no suckled control rats. It is possible that these peptides when they reach the median eminence where they are positioned for release into the portal circulation are rapidly released to the portal vessels for the transport of these peptides to the pituitary.

Alternatively, these peptides may inhibit tuberoinfundibular dopaminergic neuron activity, which could be an additional mechanism to generate enhanced PRL secretion (46). Although the presence of binding sites for pFE22 in the anterior pituitary gland has yet to be reported, this would be evidence to support the release of this peptide into the portal circulation and direct actions on the pituitary. A pituitary site of action is also suggested by a recent study by McGivern et al. (14), where a systemic injection of pFE22 inhibits stress-induced ACTH, corticosterone, and PRL secretion. However, an intracerebroventricular injection of this peptide is without effect on hormone release, despite anxiolytic behavioral effects. The stimulatory effects of pFE22 on PRL release observed in the present study appear to be contrary to the inhibitory effects of this peptide on restraint stress-induced PRL release. However, the markedly different experimental situations, i.e. in vitro incubation and in vivo suckled female rats vs. the restrained male rat preparation used by McGivern et al. (14) may account for this difference. Further, McGivern et al. (47) also report that these peptides stimulate PRL secretion from superfused pituitary cells in vitro confirming the presently reported effects. Although the role of TRH in the regulation of suckling-induced PRL secretion has been proposed (38, 48, 49), this effect has not been replicated by others (50), so the exact role of TRH in suckling stimulated PRL secretion has been unclear. However, what is clear is that neurons in the parvocellular portion of the PVN are involved in suckling-induced hormone release because ample numbers of studies report that suckling induces c-fos expression (protein and mRNA) in the PVN (51, 52). It would appear that nipple stimulation, and eventually, milk letdown activate significant populations of PVN neurons, which may include the PVN neurons containing preproTRH.


   Acknowledgments
 
We would like to thank Dr. Iris Lindberg for providing the PC2 enzyme. We would also like to thank Virginia Hovanesian for her imaging work.


   Footnotes
 
1 This work was supported by the National Science Foundation (Grant No. IBN-9507952 to E.A.N.). Back

Received July 6, 2000.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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