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Endocrinology, doi:10.1210/en.2003-0829
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Endocrinology Vol. 145, No. 2 519-528
Copyright © 2004 by The Endocrine Society

Biosynthesis of Proopiomelanocortin-Derived Peptides in Prohormone Convertase 2 and 7B2 Null Mice

Virginie Laurent, Lisa Jaubert-Miazza, Roxane Desjardins, Robert Day and Iris Lindberg

Department of Biochemistry and Molecular Biology (V.L., L.J.-M., I.L.), Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112; and Département de Pharmacologie (R.De., R.Da.), Faculté de Mèdecine et Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, Québec, Canada JIH 5N4

Address all correspondence and requests for reprints to: Iris Lindberg, Ph.D., Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, 1901 Perdido Street, New Orleans, Louisiana 70112. E-mail: ilindb{at}LSUHSC.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Prohormone convertases (PCs) are thought to represent the major proteinases involved in the biosynthetic processing of peptide hormone precursors to bioactive peptide products. The maturation of PC2 requires the aid of a helper protein, 7B2, in order for the zymogen to become an active enzyme species. The 7B2 and PC2 nulls should thus be functionally equivalent with regard to deficits in precursor processing. In this article, we have examined this proposition through the study of proopiomelanocortin (POMC) biosynthesis and granule content in both null models. RIA data indicate that both PC2 and 7B2 nulls lack pituitary {alpha}-MSH; interestingly, 7B2 nulls are still able to generate ß-endorphin from ß-lipotropin, whereas PC2 nulls contain little if any ß-endorphin. Labeling experiments demonstrate a build-up of POMC, high molecular weight intermediates, and intact ACTH, as well as the disappearance of {alpha}-MSH, in both null models. Electron microscopy of neurointermediate lobe melanotrophs reveals the presence of a significantly greater number of secretory granules in both 7B2 and PC2 nulls compared with wild-type controls. However, PC2 null melanotrophs contain twice as many granules as 7B2 null melanotrophs. Another difference between the two null models is a relatively enhanced accumulation of precursors in the PC2 null compared with the 7B2 null; these include not only PC2 substrates, but also presumed PC1 substrates. These data indicate that the two nulls are not phenotypically equivalent.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROOPIOMELANOCORTIN (POMC) is the precursor to ACTH as well as to other bioactive peptide hormones, such as the opioid peptide ß-endorphin, and {alpha}-MSH, which plays an active role in skin pigmentation (reviewed in Ref. 1). The POMC precursor is expressed in the corticotroph cell population of the anterior pituitary but is also expressed in melanotrophs, which constitute the major cell population of the intermediate pituitary.

POMC is a 30–32 kDa molecule (2, 3) composed of three main regions: an N-terminal {gamma}-MSH-containing sequence, the central ACTH (1–39) sequence (with the {alpha}-MSH precursor as its N terminus), and the C-terminal ß-lipotropin (LPH) sequence, which can be processed into {gamma}-LPH and ß-endorphin. In common with other peptide hormone precursors, such as proenkephalin (4) or the common precursor to arginine vasopressin and neurophysin II (5), the proteolytic cleavage of POMC is regulated in a tissue- and cell-specific manner (6, 7, 8).

POMC has long been studied as a model for proneuropeptide and peptide hormone processing by convertases (reviewed in Ref. 7). Briefly, in the anterior pituitary, the subtilisin-like prohormone convertase (PC) 1 cleaves POMC to yield ACTH and ß-LPH. In the neurointermediate lobe and in the central nervous system, PC2 generates {alpha}-MSH, corticotropin-like intermediate lobe peptide (CLIP), {gamma}-LPH, and ß-endorphin (reviewed in Ref. 7). As a result, depending on the profile of convertases present, the POMC precursor can give rise to various bioactive peptides that have a wide spectrum of actions throughout the body.

Data from our laboratory have shown that pro-PC2 must interact with the small neuroendocrine protein 7B2 in the secretory pathway to generate an active PC2 molecule (9, 10). Paradoxically, 7B2 also acts to oppose PC2 action because both full-length 7B2 as well as the 31 residue carboxyl-terminal domain represent inhibitors of active PC2 (11, 12); however, in vivo, the facilitatory action of 7B2 on PC2 appears to be dominant (reviewed in Ref. 13). 7B2 does not affect PC1 or furin activities and does not coimmunoprecipitate with other convertases (14), suggesting that it is a specific binding protein for PC2. The physiology of the 7B2 and PC2 genes has recently been investigated with the creation of mice null for either PC2 or 7B2 (15, 16). Although PC2 nulls display distinct metabolic defects, such as a complete lack of glucagon, hypoglycemia, and altered CNS opioid peptide levels (17, 18), they exhibit a normal lifespan. In contrast, although they share some of the same symptoms as the PC2 nulls, 7B2 nulls develop a lethal form of Cushing’s disease due to excess ACTH secretion and die at about 5 wk of age (16, 19). In both 7B2 and PC2 null mice, the loss of PC2 activity results in very high levels of intact intermediate-lobe ACTH (16, 20). Interestingly, this stored ACTH is released into the circulation in much greater quantities in the 7B2 null than in the PC2 null. Indeed, circulating ACTH levels can reach 3 ng/ml in the 7B2 null, a level 60-fold higher than the normal wild-type (WT) level. The different backgrounds of the two nulls are likely to play a role in these different phenotypes, because the PC2 null was created on a 50:50 mix of 129/Sv and C57Bl/6J, whereas the 7B2 null is on a complete 129/Sv background.

In the study described here, we have examined the biosynthesis and processing of the POMC precursor and POMC-derived hormones in 7B2 and PC2 nulls compared with WT mice. We have analyzed the pituitary contents of ACTH, {alpha}-MSH, and ß-endorphin using RIAs with specific antisera. We performed steady labeling of null and control neurointermediate pituitaries as well as quantitative real-time PCR to study possible alterations in POMC synthesis among the different genotypes. Lastly, we have examined the granule content and morphology of the different nulls and controls through electron microscopy. Our data indicate that, counter to expectations, the two nulls are not phenotypically identical.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
7B2 129/Sv and PC2 129/Sv-C57BL/6J WT and null mice were obtained by breeding 7B2 and PC2 heterozygote animals. The original 7B2 null mutant mice were generated by embryonic stem-targeting technology via a transposon-based system (21, 22). The PC2 null mutant mouse line was generated as previously described (15). The colonies were maintained in an Association for Assessment and Accreditation of Laboratory Animal Care-approved facility in cages containing one to three animals of the same age (within 1 wk), sex, and genotype. 7B2 null animals used in this study had been maintained in our colony for seven to 12 generations removed from the founder animals created by the Leder laboratory in 1998; PC2 null animals used were maintained for five to seven generations after receipt from the Steiner laboratory in 1999. Animals were fed a LabDiet (Richmond, IN) 50:15 mouse chow containing 11% fat. Animals were always used at approximately 5 wk of age; data from males and females were analyzed separately (except in pituitary POMC Western blot experiments in which differences observed were clearly not related to sex but to genotype). All protocols were approved by the Louisiana State University Health Sciences Center Animal Care Committee.

Analysis of whole pituitary ACTH, ß-endorphin, and {alpha}-MSH
Two age- and sex-matched pairs of 5-wk-old 7B2 or PC2 WT and null mice were assayed independently for each POMC-derived peptide by RIA. Pituitaries from WT and null animals of the same sex and age were removed and individually collected in 1.5-ml tubes. Each pituitary was homogenized via sonication (setting 4, 10 sec) in 150 µl of ice-cold 1 N acetic acid. The samples were stored at -80 C before chromatographic separation. After thawing on ice, the samples were centrifuged for 15 min at 13,000 rpm (17,383 x g) at 4 C. The clear supernatants were individually harvested into fresh tubes. One hundred microliter aliquots of each supernatant were fractionated by high-pressure gel permeation chromatography (HPGPC) into 50 0.5-ml (1 min) samples, as described previously (16). Carrier protein, BSA (50 µl of a 0.1 mg/ml solution of crystalline BSA) was immediately added to tubes containing POMC-derived peptides. The HPGPC fractions were frozen at -20 C before analysis by the mini-RIA method (23). Briefly, in this method, samples are handled together in a 96-tube rack throughout the RIA procedure. Ten microliters of either pure (ACTH assay) or 20-fold diluted fractions ({alpha}-MSH and ß-endorphin) were subjected to assay in duplicate; dilutions were performed in RIA buffer [100 mM sodium phosphate (pH 7.4), containing 0.1% heat-treated BSA, 50 mM NaCl, and 0.1% sodium azide]. The polyclonal anti-ACTH antiserum Kathy (a gift from Dr. R. E. Mains, University of Connecticut, Storrs, CT), which recognizes both ACTH and CLIP, was used at a working dilution of 1:8000. The anti-{alpha}-MSH antiserum was a commercial sheep {alpha}-MSH polyclonal antibody from Chemicon (catalog no. AB5087; Temecula, CA) diluted to a working concentration of 1:7000. ß-endorphin was detected using the polyclonal antiserum Bunny 3 (provided by Dr. Robert Dores, University of Denver, Denver, CO) at a 1:2000 working dilution. 125I labeling of ß-endorphin and {alpha}-MSH was achieved with the chloramine T method, whereas 125I-ACTH was purchased from Amersham Pharmacia Biotech (Uppsala, Sweden).

Metabolic labeling of 7B2 null and WT intermediate pituitaries
7B2 null and WT mice were killed by rapid decapitation, and their pituitaries were removed and dissected into anterior and neurointermediate lobes in cold PBS under a stereomicroscope. The neurointermediate lobes were individually collected into 1.5-ml tubes each containing 0.5 ml of well-gassed DMEM (ICN, Aurora, OH)/BSA [0.2% fatty acid-free BSA, fraction V, Roche Diagnostics, Basel, Switzerland; 20 mM HEPES (pH 7.2)] lacking methionine. The medium was then carefully removed from each tube and replaced with 0.25 ml of 0.5 mCi/pituitary 35S-methionine in methionine-free DMEM/BSA. Oxygen was overlaid on the medium in the tubes, which were capped and placed in a shaking bath at 37 C for 3 h. The labeling medium was replaced by a fresh supply containing another 0.5 mCi of radioactive methionine halfway through the incubation. After the labeling, the tissues were quickly washed twice with 0.5 ml of gassed chase medium (regular high-glucose DMEM containing normal levels of methionine and 0.2% BSA) and were incubated in 0.25 ml of chase medium for 2 h in a shaking bath at 37 C. The chase medium was then harvested and gently centrifuged to pellet any detached cells. Supernatants were recovered into fresh microfuge tubes and supplemented with Triton X-100, Nonidet P-40, and EDTA to mimic the AG buffer concentrations and conditions used with tissues. The labeled pituitaries were each homogenized in 0.25 ml of ice-cold 5 N acetic acid/0.2% BSA by brief sonication on ice (setting 3, 5 sec) and frozen on dry ice. Upon thawing, samples were centrifuged for 10 min at 14,000 rpm at 4 C. The supernatants were lyophilized overnight and resuspended in 150 µl of AG buffer [100 mM sodium phosphate buffer (pH 7.4), 1 mM EDTA, 0.1% Triton X-100, 0.5% Nonidet P-40, and 0.9% NaCl] for immunoprecipitation. Steady labeling with 35S-methionine was performed similarly, but the tissues were incubated for 6 h in labeling medium (with fresh medium at 3 h) and were not chased. All experiments were repeated at least twice for both genotypes and sexes.

Immunoprecipitation
Immunoprecipitation of the tissue extracts as well as the chase media was conducted using either polyclonal rabbit antiserum LSU41, which is directed against the N-terminal portion of ACTH (residues 1–18, coupled to keyhole limpet hemocyanin), or JH2, which is directed to ß-endorphin (a gift of Dr. R. E. Mains). One hundred microliters of 20% prehydrated protein A-Sepharose CL-4B beads in AG buffer (Amersham Pharmacia Biotech) were added to each 150 µl of AG buffer-reconstituted extract as well as to 0.25 ml of media samples. The samples were precleared for 1 h at 4 C and centrifuged for 5 min at 14,000 rpm at 4 C in a microcentrifuge. The supernatants were individually collected into fresh tubes and were either stored frozen or used immediately for immunoprecipitation. Generally, 50 µl of the tissue samples and 100 µl of the media samples were immediately pipetted into fresh tubes. The precleared tissue extracts were diluted with 150 µl of AG buffer and 100 µg of purified LSU41 Ig (prepared by carboxymethyl cellulose affinity purification followed by ammonium sulfate precipitation), and 5 µl of stock p-chloromercuriphenyl sulfonic acid (10 mM) and 5 µl of stock phenylmethylsulfonylfluoride in ethanol (100 mM) were added to both the tissues and media tubes. Samples were incubated for 5 h at 4 C with constant rocking. One hundred microliters of a 20% solution of protein A-Sepharose CL-4B in AG buffer were then added, and the samples were rocked for 1 h at 4 C. Samples were then centrifuged for 5 min, and the beads were washed three times with 1 ml of ice-cold AG buffer, two times with 1 ml of cold 0.5 M NaCl in PBS, and four times with 1 ml of cold PBS alone. Immunoprecipitated proteins were extracted from the beads by adding 150 µl of the HPGPC eluant (but also containing 6 M urea and 1 N acetic acid) and incubated at room temperature for 15 min, followed by a 5-min centrifugation at 1500 x g. Supernatants were either frozen at -80 C, or 100 µl was immediately size-fractionated by HPGPC as described previously (16).

HPGPC
The HPGPC system consisted of a TSK-GEL SW guard column (7.5 x 7.5 mm; TosoHaas, Montgomeryville, PA) and two HPGPC columns connected in series, a Protein Pak SW 300 column (300 x 7.8 mm; Waters, Milford, MA), and a Bio-Sil TSK-125 column (300 x 7.5 mm, Bio-Rad Laboratories, Hercules, CA). The radioactivity in the HPGPC flow-through was detected by online liquid scintillation spectroscopy using a b-RAM Flow-Through System, model 2 (IN/US Systems, Inc., Tampa, FL). The flow rate of the eluant (32% acetonitrile, 0.1% trifluoroacetic acid) was 0.5 ml/min, whereas the flow rate of the scintillation fluid was 4 ml/min.

RNA samples
Four 7B2 and PC2 mice of both sexes and genotypes (null and WT) were used for this experiment. Pituitaries were harvested in 0.25 ml each of RNAlater solution (Ambion, Austin, TX). Pituitaries were either kept at 4 C or frozen after 24 h in the RNAlater solution. To isolate total RNA from those tissues, pituitaries were first weighed, individually transferred to fresh tubes, and covered with 100 µl of Lysis/Binding solution from the RNAqueous kit (Ambion). The tissues were sonicated in this solution, on ice, for 10 sec at setting 4 (Sonic Dismembrator 550; Fisher Scientific, Hampton, NH). Total RNA was then prepared according to the manufacturer’s instructions (Ambion); these RNA preparations were evaluated both by UV absorbance and denaturing agarose gel electrophoresis (as per the manufacturer’s instructions). The RNA marker used was the Millenium Markers set from Ambion. RNA samples were stored at -20 C or -80 C in the elution buffer.

Real-time quantitative RT-PCR
Primers and the TaqMan probe were designed using Primer Express Software from Applied Biosystems (Foster City, CA); the amplicons and the TaqMan probe spanned the exon 1/exon 2 splice site for POMC mRNA. The TaqMan probe (5'-AGCTGCCTTTCCGCGACAGGG-3') was labeled with 6FAM (6-carboxyfluorescein) at the 5' end and contained the quencher TAMRA (6-carboxy-N,N,N',N'-tetramethylrhodamine) at the 3' end (Applied Biosystems). The forward primer sequence was 5'-AGGCCACTGAACATCTTTGTCC-3', and the reverse primer was 5'-GGCAAACAAGATTGGAGGGA-3' (Integrated DNA Technologies, Inc., Coralville, IA). Predeveloped TaqMan Assay Reagents for measuring 18S rRNA were purchased from Applied Biosystems. RT-PCR assays were set up using the One-Step RT-PCR kit (Applied Biosystems). Each POMC reaction contained 5 µl (5 ng) RNA, 900 nM forward primer, 900 nM reverse primer, 100 nM TaqMan probe, 1x Master mix and Multiscribe enzyme mix from the One-Step RT-PCR, and diethylpyrocarbonate-treated H2O up to a total volume of 25 µl per reaction. Each control reaction measuring 18S rRNA contained 5 µl RNA sample, 1x primers and probe mix from the Predeveloped TaqMan Assay Reagents kit, 1x Master Mix and Multiscribe enzyme mix from the One-Step RT-PCR kit, and diethylpyrocarbonate-treated H2O up to 25 µl. The assays were conducted on an ABI Prism 7700 Sequence Detection System from Applied Biosystems at the following thermal cycling parameters: 30 min at 48 C, 10 min at 95 C, 40 cycles of 15 sec at 95 C, and 1 min at 60 C. Amplification curves were visually inspected to set a suitable baseline range and threshold level. The fractional number of PCR cycles required to reach the threshold fluorescence level was scored and used for generating standard curves and interpolating RNA concentration levels.

SDS-PAGE and Western blotting analysis of pituitary POMC
Pituitaries used in this experiment were harvested from 5-wk-old animals and stored frozen at -80 C. Four 7B2 and PC2 null and WT pituitaries derived from both sexes (a total of 32 unique samples) were individually sonicated (10 sec at setting 4) in 250 µl ice-cold Laemmli sample buffer [50 mM Tris/HCl (pH 6.8), 2% sodium dodecyl sulfate, 10% glycerol, 5% ß-mercaptoethanol, and 0.6% bromophenol blue] with 6 M urea. The pituitary extracts were stored frozen in 40-µl aliquots at -20 C before SDS-PAGE and Western blotting analysis. Samples were boiled 5 min and centrifuged briefly to collect the liquid at the bottom of the tubes. Proteins were separated on 8.8% (tubulin) or 15% (POMC) gels, transferred to nitrocellulose (Bio-Rad), and blocked in 1% BSA in Tris-buffered saline [50 mM Tris-HCl buffer (pH 7.4), 200 mM NaCl] before antiserum recognizing POMC (Bunny 2, used at 1:1000; a gift of Dr. Robert Dores) incubation or before the addition of the tubulin monoclonal antibody (Zymed, South San Francisco, CA; catalog no. 13-8000; used at 1:1000). The membranes were incubated in their respective primary antibody overnight at 4 C and then washed four times (5 min each) with 20 mM Tris buffer (pH 7.6) containing 0.8% NaCl and 0.8% Tween 20. The washing steps were followed by 1 h of incubation in the secondary antibody solutions (1:10,000 for all antibodies), which were either horseradish peroxidase-conjugated goat antimouse antibody (Immunopure; Pierce Chemical Co., Rockford, IL) for tubulin or a horseradish peroxidase-conjugated goat antirabbit antibody (Immunopure; Pierce) for POMC. After washing the membranes extensively, blots were incubated for 1 min in a 1:1 mixture of the Supersignal West Pico Peroxide solution (Pierce) and the Luminol/Enhancer solution (Pierce). The membranes were then wrapped in a plastic sheet, and the chemiluminescent bands were revealed using the Fluor-S Max MultiImager System from Bio-Rad. The densities and volumes of bands were calculated using QuantityOne software (Bio-Rad) for tubulin normalization.

Electron microscopy
The preparation of tissues for electron microscopy was carried out as previously described (24). Briefly, pituitaries from animals of different genotypes were fixed in 2.5% glutaraldehyde in 0.05 M sodium cacodylate buffer (pH 7.4). After fixation, the tissues were transferred to a fresh solution of 1% osmium tetroxide for 1 h at 4 C and then to 0.25% uranyl acetate in sodium acetate buffer (pH 6.3) for 45 min at 4 C. After an overnight rinse in 0.25% uranyl acetate (in water), the tissues were dehydrated in increasing concentrations of acetone and then embedded in Epon. The tissue sections were stained with 1% uranyl acetate and 6% lead citrate (pH 10). Quantitation was carried out on two to four pituitaries. For each pituitary, two pictures of identical surface areas were taken at a magnification of x7000. Granules were counted manually. Two different areas of melanotrophs were quantified per pituitary, and the mean of these values per unit of surface area is reported in Table 1Go. Data for males and females are given. Statistical analyses were carried out using the Student’s t test, where P < 0.05 is considered significantly different.


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  		TABLE 1. Number of granules in melanotrophs from mice of different genotypes

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Whole 7B2 and PC2 null pituitaries contain ACTH and ß-endorphin but no {alpha}-MSH
To study the processing of the POMC precursor in 7B2 and PC2 null pituitaries, we performed RIAs using specific antisera directed against POMC-derived peptides. Pituitaries from two pairs of PC2 and 7B2 null and WT animals were homogenized, and acid-soluble peptides were fractionated using HPGPC. We then performed RIAs using anti-{alpha}-MSH and anti-ß-endorphin antisera. Similar to what was found in earlier work (16), our ACTH assays showed that the 7B2 null pituitary contains extremely elevated levels of ACTH. The ACTH antiserum used here cross-reacts with processed forms such as CLIP. We observed ACTH, but no CLIP, in the pituitaries of 7B2 nulls (Fig. 1AGo). Interestingly, RIA of the same samples for ß-endorphin-immunoreactive (ir) peptides showed that 7B2 null pituitaries yielded as much ß-endorphin-ir as 7B2 WT pituitaries (Fig. 1BGo), an unexpected finding because it has been shown that ß-endorphin cleavage from ß-LPH is mediated by PC2 in the mouse neurointermediate lobe (25). Further processing of ß-endorphin to its truncated form ß-endorphin (1–27) was also blocked in the 7B2 null compared with WT animals [compare the retention time of the major ß-endorphin-ir peak, fraction 35, vs. the known retention time of the N-acetyl ß-endorphin (1–27) standard, fraction 38]. Figure 1CGo confirms the abolition of {alpha}-MSH production in the 7B2 null with a concomitant increase in intact ACTH (detectable due to apparent cross-reaction of the anti-{alpha}-MSH antiserum).



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  		FIG. 1. Pituitaries of 7B2 null mice exhibit a high ACTH level, a normal level of ß-endorphin, but no {alpha}-MSH. Two 7B2 and PC2 null and WT pituitaries from both sexes were individually collected for RIA analysis using the following three specific antisera: anti-ACTH antiserum Kathy (directed against the C-terminal portion of ACTH); anti-ß-endorphin antiserum Bunny 3, and anti-{alpha}-MSH antiserum. A, B, and C, ACTH, ß-endorphin, and {alpha}-MSH RIAs, respectively, of size-fractionated acid extracts. The concentration of peptides is expressed as pmol per pituitary; the experiments were repeated on two to three independent sets of pituitaries.

 
ACTH, {alpha}-MSH, and ß-endorphin RIAs were also performed using PC2 null pituitaries. Figure 2AGo shows that, like the 7B2 null, the PC2 null pituitary accumulated a dramatically high level of intact ACTH. In contrast to the 7B2 null, however, the conversion of ß-LPH into ß-endorphin and {gamma}-LPH was nearly abolished (Fig. 2BGo). Because little or no ß-endorphin was produced in the PC2 null, the occurrence of further processing to the truncated form ß-endorphin (1–27) could not be examined. Figure 1CGo depicts the profile of {alpha}-MSH-ir peptides, which supports the idea that {alpha}-MSH-sized peptides are absent in the PC2 null.



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  		FIG. 2. The PC2 null pituitary exhibits a high level of ACTH and ß-endorphin and no {alpha}-MSH. POMC processing in PC2 null and WT mice was analyzed by RIA using the same antisera as in Figure 1Go. A, ACTH RIA. B and C, ß-endorphin and {alpha}-MSH RIAs, respectively. Note that the PC2 WT pituitary exhibits a minor product in fraction 38 that corresponds to the position of the N-acetyl-ß-endorphin (1–27) standard. D, Radiolabeled ß-endorphin-ir, ß-LPH-ir, and POMC-ir molecules are depicted after separation on a 15% SDS-PAGE gel. Metabolic labeling of POMC-derived peptides from 7B2 nulls, PC2 nulls, and a control neurointermediate pituitary was carried out for 3 h, followed by a 2-h chase; antiserum JH2 was used. KO, Knockout.

 
To confirm the lack of ß-endorphin production in the PC2 null, metabolic labeling of POMC-derived peptides was performed using neurointermediate lobes from both null models (Fig. 2DGo). Pituitaries were labeled for 3 h with radioactive methionine and chased for 2 h; extracts were then immunoprecipitated with anti-ß-endorphin antiserum. Radiolabeled ß-endorphin-ir, ß-LPH-ir, and POMC-ir peptides were visualized after separation on a 15% SDS-PAGE gel, drying, and phosphoimaging. The WT control sample exhibited a single large band corresponding to ß-endorphin (lane 1). The 7B2 null contained peptides corresponding to mature ß-endorphin and also ß-LPH- and POMC-sized molecules (lane 2). However, PC2 null pituitary ß-endorphin-immunoprecipitable products were almost completely restricted to a ß-LPH-sized peptide (lane 3). Although a faint band corresponding to the mass of ß-endorphin still persisted, the cleavage to mature ß-endorphin was apparently well blocked in the PC2 null. These results, consistent with the RIA data, support the finding that there are unexpected differences in ß-endorphin production between the 7B2 and PC2 nulls.

POMC biosynthesis in 7B2 and PC2 null pituitaries
Metabolic labeling of POMC, using 7B2 and PC2 null and WT pituitaries, was performed to assess whether alterations in POMC biosynthesis could contribute to the extremely high levels of pituitary ACTH in both null models. Previous studies have shown that the cleavage of ACTH to {alpha}-MSH does not occur in 7B2 nulls; high molecular weight (HMW) forms of ACTH and ACTH itself persist during the chase period (16). Confirming these results, metabolic labeling of POMC and POMC-derived peptides illustrates a complete lack of {alpha}-MSH production in the 7B2 null neurointermediate pituitary in the face of accumulation of both intact ACTH and an HMW intermediate (HMWI, Fig. 3AGo, panel 4). 7B2 WT tissue and medium samples both contained only the N-terminal peptide of ACTH processing in the intermediate pituitary, {alpha}-MSH. Metabolic labeling of POMC was also carried out using PC2 null and WT neurointermediate lobes (Fig. 3BGo). After a 3-h pulse, PC2 null pituitaries contained immunoprecipitable radiolabeled POMC, HMWI, and ACTH, but no {alpha}-MSH-ir materials. PC2 WT tissue and medium chased for 2 h in the presence of nonradioactive methionine both contained a single {alpha}-MSH-sized labeled molecule (Fig. 3BGo, panels 1 and 2, respectively). However, in PC2 nulls, POMC, HMWI, and ACTH were the major immunoprecipitable species (Fig. 3BGo, panel 3). This pattern persisted in the PC2 null chase medium, which also contained POMC, HMWI, and ACTH (Fig. 3BGo, panel 4). We observed a greater quantity of POMC and HMWIs in the PC2 null pituitary than in the 7B2 null pituitary. These results showing the persistence of intermediates during the chase period in the PC2 null resemble, but are more exaggerated than, the results found in the 7B2 null (16).



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  		FIG. 3. Both PC2 and 7B2 null pituitaries exhibit increased amounts of HMW precursors compared with WT controls. Freshly dissected neurointermediate pituitaries from PC2 and 7B2 null and WT animals were collected into methionine-free medium, labeled for 3 h, and chased for an additional 2 h, as described in Materials and Methods. Immunoprecipitation of various POMC-derived peptides was performed on chased tissue extracts and on chase media using purified anti-ACTH IgG. A and B, The profile of size-fractionated radiolabeled peptides in immunoprecipitates from chased tissue and medium, respectively. Panels A1 and A2 illustrate 7B2 WT chased tissue and medium, respectively, whereas panels A3 and A4 represent 7B2 null chased tissue and medium, respectively. Panels B1 and B2 depict PC2 WT chased tissue and medium, respectively. Panels B3 and B4 show PC2 null chased tissue and medium, respectively.

 
In addition to metabolic labeling and RIA, the content of POMC-derived peptides in 7B2 and PC2 neurointermediate pituitaries was studied using 35S-methionine steady labeling. Three 7B2 null and three control WT neurointermediate lobes were each labeled for 6 h before immunoprecipitation using LSU41 antiserum. 7B2 WT tissues displayed a single peak corresponding to {alpha}-MSH (Fig. 4AGo). In contrast, 7B2 null tissues contained radiolabeled POMC, HMWI, and high levels of ACTH, but no {alpha}-MSH-sized peptides (Fig. 4BGo). The same experiment was carried out with two sets of PC2 WT and PC2 null pituitaries. Figure 4CGo depicts the results obtained using PC2 WT neurointermediate lobe, which contained only POMC and {alpha}-MSH-sized peptides. In contrast, PC2 null neurointermediate lobes contained POMC, HMWI, and ACTH, but no {alpha}-MSH-sized molecules (Fig. 4DGo). These steady labeling experiments complement the RIA data by showing that both PC2 and 7B2 nulls accumulate not only ACTH, but also HMWI thought to be cleaved by PC1.



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  		FIG. 4. Steady labeling of POMC-derived peptides in the 7B2 and PC2 null neurointermediate lobes shows increased levels of HMW POMC intermediates compared with WT. Neurointermediate lobes from 7B2 null and control WT mice were labeled for 6 h before immunoprecipitation of POMC and POMC-derived proteins, size fractionation, and online scintillation spectroscopy. A and B, Steady labeling of POMC-derived peptides from 7B2 WT (A) and 7B2 null (B) neurointermediate lobes. C, PC2 WT neurointermediate lobe POMC immunoprecipitates. D, Immunoprecipitates from PC2 nulls.

 
Total POMC mRNA levels are decreased in the PC2 null but not in the 7B2 null pituitary
Pituitary mRNA analysis was performed to compare the expression of POMC mRNA in 7B2 and PC2 nulls. Four whole pituitaries of each genotype (7B2 and PC2 null and each WT) and each sex were individually collected, and total RNA was extracted from each. Analysis of POMC mRNA was performed using real-time quantitative RT-PCR with normalization of input RNA to the 18S RNA control. Results are given as the relative POMC concentration, where 200 U on the y-axis is equivalent to 3.1 µg/ml of POMC. The same 7B2 WT male sample was always used as an internal standard. POMC mRNA levels were found to be similar in the 7B2 null and WT animals (Fig. 5Go). By contrast, POMC mRNA levels in PC2 null males were significantly decreased compared with their WT controls, and POMC mRNA levels were slightly lower in the PC2 null females compared with controls. These data indicate that POMC mRNA expression is apparently somewhat decreased in the PC2, but not the 7B2, null pituitary.



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  		FIG. 5. Total POMC mRNA levels are unchanged in the 7B2 null but are decreased in the PC2 null pituitary. Pituitaries from four 7B2 or PC2 male (M) and/or female (F) nulls (labeled as 7B2 KO and PC2 KO) and an equivalent number of WT male and female pituitaries were individually collected before extraction of total RNA. Normalization of the mRNA level of each pituitary was obtained using real-time quantitative RT-PCR analysis of 18S RNA. The real-time RT-PCR experiment for POMC mRNA quantitation was performed three times using a 7B2 WT male sample as an internal standard. Results are shown as the relative concentration of POMC mRNA to the standard. On the y-axis, 200 represents 3.12 µg/ml total RNA. Data are represented as the mean ± SD; n = 4 pituitaries of each sex and each genotype. The Student’s t test was used to compare PC2 WT and KO; ***, P < 0.0004. KO, Knockout.

 
PC2 null pituitaries exhibit a higher content of POMC than the 7B2 null and WT controls
Age-matched 7B2, PC2 null, and WT pituitaries of both sexes were analyzed for their POMC contents using SDS-PAGE followed by Western blotting with anti-POMC and antitubulin antisera (for normalization of samples) in four separate experiments. Because similar differences between genotypes were observed both for males and for females, data for both sexes were pooled. PC2 null pituitaries exhibited a higher level of POMC than their WT controls; in contrast, 7B2 null POMC levels were reduced compared with WT controls (Fig. 6Go). Both the glycosylated and unglycosylated forms of POMC were similarly increased in null pituitaries (data not shown). Interestingly, the POMC content of the 7B2 null pituitary was significantly lower than the POMC content of the PC2 null pituitary. Taken together with the RIA data presented in Figures 1Go and 2Go, these data suggest that the decreased message levels of POMC in the PC2 pituitary do not result in decreased protein levels and that POMC levels are actually higher in the PC2 null than in the 7B2 null, which is in keeping with the higher tissue content of ACTH in the former null (20).



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  		FIG. 6. Pituitary POMC protein levels in PC2, but not 7B2, nulls are higher than in WT controls. Results are presented as a ratio of arbitrary density units within the POMC band to those within the tubulin band (for normalization of the protein quantity in each sample). Results are shown as the mean ± SD (n = 5). **, P < 0.005 (between the 7B2 null and the PC2 KO); ***, P = 0.0006 (PC2 KO compared with the PC2 WT). KO, Knockout.

 
7B2 and PC2 null pituitaries contain more granules than their WT controls; PC2 null intermediate lobes contain more granules than 7B2 null intermediate lobes
To investigate whether pituitary ACTH and POMC could be differentially stored in the various mutant mice, we examined the morphology of 7B2, PC2 null, and WT control intermediate lobes using electron microscopy (Fig. 7Go). 7B2 and PC2 null intermediate lobes (Fig. 7Go, B and D, respectively) both exhibited many more secretory granules than the 7B2 and PC2 WT pituitaries (Fig. 7Go, A and C, respectively). This finding is in agreement with the high levels of ACTH detected in the intermediate lobes of both null mutants (16, 20). In addition, neurointermediate lobes from PC2 nulls appear to contain twice as many granules as 7B2 nulls (Table 1Go), which is consistent with the higher quantities of POMC and ACTH stored in the PC2 nulls vs. 7B2 nulls, as shown in this study and elsewhere (20). Thus, increased ACTH (and POMC) content is correlated with increased numbers of granules in both null models, with the PC2 null having the highest density of secretory granules.



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  		FIG. 7. 7B2 and PC2 null intermediate lobes have a greater number of secretory granules than WT controls. A and B, Secretory granule content of 7B2 WT and null neurointermediate lobes. C, Granules within the PC2 WT neurointermediate lobe. D, Granules in PC2 null intermediate lobe.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
PC2 and 7B2 null mice represent two distinct convertase-related mutants that share disrupted neurohormone processing pathways due to the common lack of PC2 activity (16, 20, 26, 27). Due to this PC2 deficiency, both null models exhibit extremely high levels of the PC2 substrate, intermediate lobe ACTH; however, the total content of pituitary ACTH is higher in the PC2 null than in the 7B2 null (16, 20). To examine the biosynthesis and/or release of POMC-derived products in the 7B2 null compared with the PC2 null, we performed RIAs and radiolabeling of pituitaries from various genotypes using specific antisera directed to POMC-derived peptides.

The production of {alpha}-MSH, derived by PC2-mediated cleavage of ACTH (7), was similar in both nulls, as assessed by RIA. Both nulls exhibited extremely high levels of pituitary ACTH and little or no {alpha}-MSH. However, ß-LPH processing differed greatly between the two null strains. 7B2 null pituitaries were still capable of yielding ample quantities of ß-endorphin, whereas the PC2 null almost completely lacked this opioid peptide. These data were confirmed using radiolabeling of ß-endorphin-ir molecules in pituitaries of both null models and agree with the finding that ß-endorphin levels are severely reduced in the brains of PC2 null mice (28). It is interesting to note that the cleavage profiles of progastrin-derived peptides also differ between 7B2 and PC2 nulls; as with ß-LPH, a greater blockade of progastrin processing is seen in the PC2 null than the 7B2 null (31). A small amount of residual PC2 activity remaining in the 7B2 null pituitary could account for the cleavage of ß-LPH in this null; however, PC2 expressed in the absence of 7B2 has been shown to be completely inactive (16). Alternatively, there may be compensatory intervention of another enzyme that yields ß-endorphin in the one null but not the other. Bloomquist et al. (29) and Zhou and Mains (30) have demonstrated that overexpression of PC1 enhances ß-endorphin production in AtT-20 cells, suggesting that this particular cleavage event can also be mediated by PC1. We have examined the possibility of differential expression of pituitary PC1 in the various nulls; however, our results indicate that PC1 expression is not altered (A. Dubey and I. Lindberg, unpublished results).

Taken together, these data support the idea that the two null models differ in peptide precursor storage and handling. As mentioned above, the 7B2 and PC2 nulls were constructed in different strains. Differences in the morphology of the intermediate lobe have been noted between the strains involved, 129/Sv and C57Bl/6J mice (32). It is quite possible that biochemical dissimilarities accompany these morphological differences, although the actual biochemical mechanism of strain-dependent processing differences is difficult to envision. However, preliminary data on processing of POMC in double PC2/7B2 knockouts indicate that strain is indeed a contributing factor to processing efficiency (our unpublished results).

Three lines of evidence indicate that POMC processing is blocked in PC2 and 7B2 nulls at both PC1- and PC2-mediated sites. Steady labeling of neurointermediate lobes confirmed that both 7B2 and PC2 nulls contained high levels of ACTH and accumulated POMC and HMWIs. Metabolic labeling of the pituitaries of 7B2 and PC2 nulls showed increased levels of POMC and HMWI in both null models, as would be expected if downstream processing is blocked; somewhat higher levels of precursors were observed in the PC2 nulls compared with the 7B2 nulls. Finally, the findings of increased precursors was substantiated with Western blotting of total pituitary POMC; however, only the PC2 null showed an increase in POMC using this method, supporting differences in cellular handling and storage of this precursor between the two nulls. The observation of buildup of PC1 substrates in PC2-deficient mice was unexpected but not without precedent; previous studies have shown a general accumulation of neuroendocrine precursors, which include presumed PC1 substrates, in PC2 nulls, for POMC (Ref. 28 and the present study), prodynorphin (33), and procholecystokinin (31).

The increase in POMC protein in intact PC2 null pituitaries is in apparent conflict with the decrease in total pituitary POMC message levels. However, the present data are limited in that they do not indicate in which lobe the decrease in POMC mRNA occurs. The opposing data for protein and mRNA can be reconciled if synthesis of protein is differentially controlled in the two lobes because, unlike the situation in the 7B2 null, POMC mRNA synthesis is maintained in the anterior lobe of the PC2 null (20). Alternatively, discrepancies between protein and message levels may have more to do with storage and release rates than with biosynthetic control. In contrast to the PC2 null, in the 7B2 null, POMC expression is restricted to the intermediate lobe (20), which is known to be under dopaminergic control (34, 35). The decrease in pituitary dopamine previously detected in the 7B2 null (20) suggests that pituitary POMC transcription should be up-regulated; however, this was not observed. A dramatic increase in POMC mRNA in the 7B2 null intermediate lobe was also not apparent in previous in situ experiments (20), although it could have been missed by the saturation of the signal. The fact that pituitary POMC protein was decreased in the 7B2 null (as demonstrated in this study) suggests that factors other than transcriptional control affect the levels of precursor in this null, which again potentially relate to storage and release rates.

Taken together, the data presented here indicate that, despite their common loss of PC2 action, the 7B2 and PC2 nulls are not identical with regard to POMC processing and storage. Perhaps the most interesting difference between the PC2 and 7B2 nulls relates to the ability of the latter, but not the former, to perform a particular cleavage event, the cleavage of ß-endorphin from ß-LPH, illustrating a complexity in precursor processing that is difficult to explain by the simple loss of PC2 activity, which should yield similar results in both nulls (as was the case for {alpha}-MSH). The relatively enhanced accumulation of precursors in the PC2 null compared with the 7B2 null includes not only PC2 substrates but also presumed PC1 substrates. Thus, the study of the phenotypic differences between the 7B2 and PC2 null models continues to provide interesting information on the regulation of peptide precursor processing.


   Acknowledgments
 
We thank Katherine Smith for assistance with RIAs; Gregory Hubbard for maintenance of the various mouse colonies; D. F. Steiner, C. H. Westphal, and P. Leder for founder mice for the PC2 and 7B2 null colonies; W. V. Vedeckis and the Louisiana State University Cancer Center for allowing us to use the ABI prism 7700 Sequence Detection System; and K. Pedersen for providing his real-time RT-PCR expertise.


   Footnotes
 
This work was supported by NIH Grants DK48703 and DA05084 (to I.L.). I.L. was supported by a Career Development Award from the National Institute on Drug Abuse. This work was also supported by the Canadian Institutes of Health Research (to R.D.). R.D. is a scholar (Chercheur Boursier Senior) of the Fonds de la Recherche en Santé du Québec.

Abbreviations: CLIP, Corticotropin-like intermediate lobe peptide; HMWI, high molecular weight intermediate; HMW, high molecular weight; HPGPC, high-pressure gel permeation chromatography; ir, immunoreactivity; LPH, lipotropin; PC, prohormone convertase; POMC, proopiomelanocortin; WT, wild-type.

Received July 3, 2003.

Accepted for publication October 14, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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