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Endocrinology Vol. 143, No. 1 254-262
Copyright © 2002 by The Endocrine Society

NEUROENDOCRINOLOGY

Gene Expression of Proprotein Convertases in Individual Rat Anterior Pituitary Cells and Their Regulation in Corticotrophs Mediated by Glucocorticoids

Weijia Dong and Robert Day1

Département de Pharmacologie, Faculté de Médecine and Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4

Address all correspondence and requests for reprints to: Robert Day, Ph.D., Department of Pharmacologie, Faculté de Médecine, Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, 3001, 12e Avenue Nord, Sherbrooke, Québec, Canada J1H 5N4. E-mail: rday{at}courrier.usherb.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The subtilisin-like proprotein convertases (SPCs) are a family of serine proteinases that process secreted proteins at single or paired basic residues. Each SPC has been localized in the rat anterior pituitary, implying their importance in precursor processing in this tissue. The cellular distribution of each SPC has not been established in each hormone-producing cell type. We used double labeling in situ hybridization histochemistry to examine the mRNA distribution of five SPCs in relation to corticotrophs, thyrotrophs, lactotrophs, gonadotrophs, and somatotrophs. Our data demonstrated that SPC expression patterns were distinct, with each SPC expressed in more than one cell type. We noted that overlapping SPC expressions were the rule rather than the exception, suggesting potential SPC redundant functions. We examined the effects of adrenalectomy on corticotroph SPC expression. Most corticotrophs expressed SPC1, SPC3, and SPC4, but few corticotrophs expressed SPC2 or SPC6. After adrenalectomy, we observed increased mRNA levels for SPC1, SPC3, SPC4, and SPC6, but not for SPC2, in POMC-positive anterior pituitary cells. These increased levels were reversed by dexamethasone treatment. These data demonstrate the plasticity of SPCs expression in corticotrophs. SPCs may be directly involved in the mammalian stress response and may be important in hypothalamic-pituitary-adrenal axis homeostasis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ENDOCRINE ROLE of hormones produced by the anterior pituitary gland is well established. Various peptides identified in the central nervous system or peripheral tissues have also been localized in the anterior pituitary (1, 2, 3, 4, 5). These peptides together with the classical hormones function in an endocrine or autocrine/paracrine manner to regulate the endocrine system. ACTH as well as many other peptides are initially synthesized as inactive precursors and are posttranslationally processed during their intracellular transport to yield the final bioactive molecules. An important step in this processing is the cleavage of precursors at the C-terminal side of specific single or paired basic amino acids by a family of subtilisin/kexin-like endoproteinases generally known as the proprotein convertases (6). During the last several years, seven distinct proprotein convertase genes have been identified in mammalian species. Although each enzyme has been independently named by their discoverers, a unified nomenclature has been proposed (7), using the term subtilisin-like proprotein convertase (SPC) which includes SPC1 (furin/PACE), SPC2 (PC2), SPC3 (PC1/PC3), SPC4 (PACE4), SPC5 (PC4), SPC6 (PC5/PC6), and SPC7 (PC7/LPC/PC8).

Previous studies showed that SPC1 (8), SPC2 and SPC3 (9), SPC4 and SPC6 (10), and SPC7 (11) were all expressed in the anterior pituitary with apparent distinct distribution patterns. However, the anterior pituitary is highly heterogeneous, with five major hormone-producing cell types, and it therefore becomes necessary to examine the cellular distribution of each SPC to have a better understanding of cell-specific functions. Colocalization studies of SPCs with hormone markers should allow us to establish how SPCs are overlaid in their anterior pituitary distribution. We have therefore characterized SPC1, SPC2, SPC3, SPC4, and SPC6 mRNA expression in individual pituitary cell populations by double label in situ hybridization. Rat GH, PRL, POMC, FSH, and TSH cRNA probes were prepared and used as markers of somatotrophs, lactotrophs, corticotrophs, gonadotrophs, and thyrotrophs.

In previous studies we demonstrated that SPC3 mRNA levels were selectively regulated by glucocorticoid feedback within parvocellular neurons of the hypothalamic paraventricular nucleus, indicating the involvement of enzyme regulation within the hypothalamic-pituitary-adrenal (HPA) axis (12). As changes in SPC expression levels could be an important mechanism in regulating the biological output of the endocrine system through processing, it was therefore of interest to study whether SPC gene expression is also regulated in pituitary corticotrophs via glucocorticoids. Thus, we examined SPC mRNA levels in corticotrophs under resting conditions or subsequent to adrenalectomy (Adx), with or without dexamethasone (Dex) replacement. The results demonstrated that glucocorticoid feedback had an important influence on the detectable basal expression of SPC1, SPC3, and SPC4. Furthermore, removal of glucocorticoid feedback revealed an induction of SPC6, but not SPC2, mRNA expression in corticotrophs. These data reinforce the idea of the role of SPCs in the stress axis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and tissue preparation
Animal experiments were performed following the recommendations of the Canadian Council of Animal Care. Male Sprague Dawley rats (250–300 g) were used throughout this study. Twenty-three rats were divided into four groups as follows: group I, sham Adx and vehicle injection (n = 5); group II, sham Adx plus Dex treatment (n = 5); group III, Adx plus Dex treatment (n = 6); and group IV, Adx plus vehicle injection (n = 7). Adx or sham Adx was performed under methofane anesthesia. After 10 d, rats in each group received either sc injections of 500 mg/kg Dex (Sigma, St. Louis, MO) or vehicle injections twice daily for 4 d. Saline was given to all animals as drinking water after the operation. The animals were killed by decapitation. Whole pituitaries were rapidly removed and frozen in isopentane precooled to -35 C. The pituitaries were stored at -80 C and later sectioned on a cryostat at a thickness of 10 µm. The frozen pituitary sections were thaw mounted on slides coated with polylysine and stored at -80 C until further processing.

Probe synthesis
[35S]CTP- and [35S]UTP-labeled cRNA probes were prepared for SPC1, SPC2, SPC3, SPC4, and SPC6 from cDNA subclones in transcription vectors. The rat (r) SPC1 cDNA consisted of 1231 nucleotides (nt) equivalent to segment 823-2053 in human (h) SPC1 (13). rSPC2 cDNA consisted of 425 nt equivalent to segment 1574–1998 in mouse (m) SPC2 (14). rSPC3 cDNA consisted of 590 nt equivalent to segment 1841–2430 in mSPC3 (15). rSPC4 cDNA consisted of 534 nt equivalent to the segment 1153–1687 of hSPC4 (16); rSPC6 cDNA consisted of 837 nt, segment 1089–1925 (17). Nonradioactive cRNA probes were prepared using digoxigenin (Dig)-11-UTP for detection of mRNAs of rGH, rTSH, rPOMC, rPRL, and rFSH from cDNA subclones in transcription vectors as previously described (18). Gonadotrophs were solely examined using an FSH probe. The rFSH ß-subunit cDNA used (880 nts, segment 1–880) was provided by Dr. R. A. Maurer (Oregon Health Sciences Center, Portland, OR) and has previously been described (19). No LH probe was used to study gondotrophs, and although the distributions of LH and FSH are known to be highly similar, they are not exactly overlapping. The rPOMC cDNA (350 nt) was a gift from Dr. J. Eberwine (University of Pennsylvania, Philadelphia, PA) and corresponds to exon III of the POMC gene. The GH, TSH, and PRL cDNAs were obtained by RT-PCR from extracted total RNA of rat anterior pituitary as previously described (9, 20). The GH cDNA (408 nt) corresponds to the region of nt 307–714 of rat GH (GenBank accession no. V01237). The TSH cDNA (301 nt) corresponds to the region of nt 12–312 of rat TSH ß-subunit (GenBank accession no. D00578). The PRL cDNA (530 nt) corresponds to the region of nt 70–679 of rat PRL (GenBank accession no. V01249).

In situ hybridization histochemistry
The in situ hybridization protocols have been described previously (18). The prehybridization treatment includes fixation in 4% phosphate-buffered formaldehyde, proteinase K treatment (0.1 µg/ml, 10 min at 37 C), and acetylation with acetic anhydride (0.25%, vol/vol) in 0.1 M triethanolamine. The cRNA probes were diluted to 33 x 103 dpm/µl and applied at 30 µl/slide. Hybridization was carried out at 55 C for 16 h. The sections were then treated with ribonuclease A (40 µg/ml) at 37 C for 45 min, followed by a successive washes in 2, 1, and 0.5x SSC for 10 min each at room temperature and in 0.1x SSC for 45 min at 60 C. Slides hybridized with radioactively labeled cRNA probe alone were dehydrated through graded ethanol and air-dried. Slides hybridized with both radioactively and Dig-labeled cRNA probes were incubated with 2% normal sheep serum and then with alkaline phosphatase-conjugated anti-Dig antibody. This was followed by overnight incubation in the dark in the chromagen solution consisting of 0.45 ml nitro blue tetrazolium (75 mg/ml in 70% dimethylformamide) and 0.35 ml 5- bromo-4-chloro-3-indolyl phosphate (50 mg/ml in dimethylformamide). After stopping the reaction, slides were quickly dehydrated in graded ethanol and air-dried. Slides were dipped in Ilford 5KD nuclear emulsion (diluted 1:1 in water) and stored at 4 C for 2–6 wk. Autoradiograms were developed in Kodak D19 (Rochester, NY) for 2 min and fixed in 30% sodium thiosulfate for 4 min.

Analysis
Observation and analysis were carried out using a Carl Zeiss Axiophot microscope (New York, NY) equipped with a Darklite Illuminator (Micro Video Instruments, Inc., Avon, MA). Colocalization photographs were taken using double exposure settings. The autoradiographic grains were first exposed under dark light illumination, thus resulting in the white grain appearance. The second exposure shows the Dig probe labeling revealed as dark immunohistochemical staining. Quantitative studies were carried out at x400 magnification by counting the grains on Dig-labeled cells. To identify positive or negative labeling, nonspecific labeling in the background and sense control sections were used as a reference. For the percentage of labeled cells in Figs. 3Go and 5Go, each bar represents data obtained by counting approximately 700–800 cells from 3 animals. For Table 1Go and Fig. 5Go, the average grain number per cell was obtained by counting grains on approximately 135 cells from 3 animals. For Table 1Go, values were adjusted to take into account differences in the specific activity of each probe. Statistic results are expressed as the mean ± SEM. Comparison of mean values was performed by ANOVA, followed by Tukey-Kramer multiple comparison test. Differences were considered significant at P < 0.05. As a negative control for in situ hybridization, radioactively labeled sense strand probes of the same size and specific activity were applied instead of antisense strand probes, and no positive labeling was encountered in the control experiments.



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  		Figure 3. Quantitative analysis of gene expression of SPCs in individual anterior pituitary cell population by dual label in situ hybridization histochemistry. Each bar represents the percentage of cells that were positively labeled by an SPC probe within an anterior pituitary cell population identified using one of the hormone marker probes. As an example, the black bars represent POMC-expressing cells. Within this defined population, 85% of POMC-expressing corticotrophs also expressed SPC1, SPC3, or SPC4, but less than 15% of this population expressed SPC2 or SPC6. Each bar was obtained by counting 700–800 cells from sections of three different animals.

 


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  		Figure 5. Quantitative analysis of SPC mRNA in corticotrophs (POMC-labeled cells) of anterior pituitary from Sham, Sham-Dex, Adx, and Adx-Dex rats. Cell and grain counting was performed as described in Fig. 1Go and Table 1Go. A dataset is shown for each SPCs including SPC1 (A), SPC3 (B), SPC4 (C), and SPC6 (D). The analysis was also carried out for SPC2, but no significant changes were observed (data not included). Statistically significant increases are indicated (*, P < 0.05; **, P < 0.001), where the comparison is made with the sham-treated group. Dex treatment alone had no significant effect on the number of cells expressing an SPC or on the level of SPC expression (determined by grain counting). However, Dex treatment completely reversed the effects of Adx.

 

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  		Table 1. Quantitative analysis of proprotein convertase mRNA levels in the rat anterior pituitary lobe

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Anatomical distribution of SPC mRNAs in the normal rat pituitary
The distribution of SPC1, SPC2, SPC3, SPC4, and SPC6 mRNAs in the normal rat pituitary is shown in Fig. 1Go and corresponds to our previously published data (9, 10, 11). Each SPC has a distinct distribution pattern, with heterogeneous expression levels within each of the pituitary lobes. All five SPCs were expressed in the anterior lobe. Of all of the SPCs examined, SPC4 had the most widespread expression pattern within the anterior lobe (Fig. 1DGo), whereas, in contrast, SPC2 was expressed in a more select population of anterior pituitary cells (Fig. 1BGo). These images suggest that different combinations of SPCs could be expressed in the distinct hormone-producing cells of the anterior lobe, possibly with overlapping patterns.



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  		Figure 1. In situ hybridization histochemistry of SPC mRNAs in rat pituitary. A, SPC1; B, SPC2; C, SPC3; D, SPC4; E, SPC6; F, representative control section with sense probe. AL, Anterior lobe; IL, intermediate lobe; NL, neural lobe. Bar, 1 mm.

 
It is noted that within the more homogenous zone of the intermediate lobe, SPC2 is abundantly expressed and colocalized with SPC1 and SPC3 mRNAs. However, the intermediate lobe melanotrophs expressed little if any SPC4 or SPC6.

Colocalization of SPCs and hormone mRNAs in the normal rat anterior pituitary
To examine the distribution of SPCs within individual anterior lobe cell populations, we used a dual labeling in situ hybridization methodology. Hormone-producing cells were detected using Dig-labeled cRNA probes (i.e. POMC, GH, FSH, PRL, and TSH), whereas SPC mRNAs were detected using radioactively labeled cRNA probes. Dig-labeled cells were revealed as a dark-staining precipitate, whereas radioactively labeled cells were detected by the presence of silver grains (i.e. observed as white dots under dark field microscopy). Representative examples of the complex labeling patterns are shown in Fig. 2Go, including SPC1 expression in lactotrophs (Fig. 2AGo), SPC3 and SPC2 in thyrotrophs (Fig. 2Go, B and C) and of SPC6 in gonadotrophs (Fig. 2FGo). For the very widely expressed SPC4 (Fig. 1DGo), we also used this methodology to demonstrate that gonadotrophs expressed less SPC4 than other anterior lobe cells (Fig. 2EGo).



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  		Figure 2. Representative examples of dual labeling in situ hybridization histochemistry of SPCs in individual anterior pituitary cell populations. The labeled SPC mRNA is represented by silver grains, which are revealed as white dots. The labeled hormone probe is represented by Dig-conjugated immunohistochemical precipitation, shown by dark staining. Colocalization of SPC1 and PRL mRNA (A), SPC3 and TSH mRNA (B), SPC2 and TSH (C), and a control section (D) labeled with sense SPC2 and Dig labeling for TSH is shown. Very few grains are observed, indicating the level of background. E, Colocalization of SPC4 and FSH mRNA. The expression levels of SPC4 were relatively lower in FSH cells than in other cell types. F, Colocalization of SPC6 and FSH mRNA. Bar, 50 µm.

 
Figure 3Go and Table 1Go summarize the complex and heterogeneous distribution of SPC mRNAs in the anterior lobe in a quantitative manner. For each anterior lobe hormone- producing cell type we have determined the percentage of cells that express a particular SPC (Fig. 3Go). For example, using POMC as a marker (Fig. 3Go, black bars), we observed that a high percentage of corticotrophs (>80%) expressed SPC1, SPC3, and SPC4, but few corticotrophs (<15%) expressed SPC2 or SPC6. We also estimated the relative abundance of each SPC within corticotrophs, as shown in Table 1Go (i.e. POMC column). The data indicate that SPC4 was clearly the most abundant SPC in corticotrophs. Although SPC2 and SPC3 mRNA levels were equally abundant in corticotrophs (Table 1Go), it should be remembered that SPC2 was expressed in very few corticotrophs (i.e. ~14%), whereas SPC3 was expressed in a large majority of corticotrophs (i.e. ~84%). For the corticotroph cell population, the sum of these data lead us to conclude that SPC4 and SPC3 are the most expressed convertases under normal conditions.

A similar analysis of each of the other cell types has been carried out. For GH-producing cells, we observed the expression of SPC1, SPC3, and SPC4 in 86–98% of all somatotrophs (Fig. 3Go). When mRNA levels were considered, SPC4 was the most abundant SPC in somatotrophs (Table 1Go). It is important to note that although SPC2 mRNA levels were slightly higher than those of SPC3 (Table 1Go), only 6% of somatotrophs express SPC2 compared with 86% for SPC3 (Fig. 3Go).

High percentages of PRL-producing cell populations were labeled with all five SPCs (Fig. 3Go); however, the highest mRNA levels were observed for SPC4, SPC2, and SPC3 (Table 1Go). In the case of thyrotrophs, high percentages of TSH-producing cells expressed SPC1, SPC2, SPC3, and SPC4, but not SPC6 (Fig. 3Go). High levels of SPC4, SPC2, and SPC3 mRNA levels were also detected in thyrotrophs (Table 1Go). It is noted that thyrotrophs expressed the highest levels of SPC2 and SPC3 compared with any other anterior pituitary cell type (see also Fig. 2Go, B and C).

Finally, all of the examined SPCs were observed in gonadotrophs. High percentages of FSH-producing cells expressed SPC1, SPC4, and SPC6 (between 78–93%), whereas SPC2 and SPC3 were expressed in fewer cells (34–54%). In terms of relative mRNA abundance, SPC4 was once again expressed at higher levels than the other SPCs in gonadotrophs (Table 1Go). Of all of the hormone-producing pituitary cell types, gonadotrophs were the major cell expressing SPC6 (Fig. 2FGo). More specifically, SPC6 was principally expressed in almost all gonadotrophs (i.e. nearly 90%) and in a subpopulation of lactotrophs (i.e. 50%; Fig. 3Go).

Effects of Adx and Dex on SPC mRNA levels in anterior pituitary corticotrophs
We next examined the regulation of each SPC within the corticotroph anterior lobe cell population under the following four conditions: 1) sham treatment, 2) sham and Dex treatment, 3) Adx treatment, and 4) Adx followed by Dex treatment. As expected, after Adx, POMC mRNA levels, were greatly increased in corticotrophs, whereas administration of Dex completely reversed these changes (data not shown). These observations confirmed the success of Adx treatment and Dex replacement. Having confirmed that the treatments were effective, we then focused our attention on the expression of SPCs within corticotrophs of these same animals. Figure 4Go shows examples of observed changes in SPC mRNA levels within corticotrophs after Adx treatment. The most important changes were seen for SPC3 (Fig. 4BGo), SPC4 (Fig. 4DGo), and SPC6 (Fig. 4FGo).



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  		Figure 4. In situ hybridization histochemistry showing the colocalization of SPCs mRNA and POMC mRNA in corticotrophs after sham control treatment or Adx. The labeled SPC mRNA is represented by silver grains, which are revealed as bright dots. The labeled hormone (POMC) probe is represented by Dig-conjugated immunohistochemical precipitation shown by the dark brown staining. A, C, and E, Representative pituitary sections from sham-treated rats; B, D, and F, sections from Adx rats. All sections are labeled for corticotrophs using POMC as a marker probe, whereas SPCs are in the following order: A and B, SPC3; C and D, SPC4; and E and F, SPC6. Bar, 50 µm.

 
To quantitatively evaluate the effects of these treatments on SPC gene expression within the corticotroph cell population, we determined both the percentage of SPC-labeled cells as well as the SPC mRNA levels using grain counting (Fig. 5Go). Significant changes were observed for SPC1, SPC3, SPC4, and SPC6. We did not observe any significant differences for SPC2 under any of the four treatment conditions (data not shown). The greatest increases observed in the percentage of labeled corticotrophs were for SPC6 (Fig. 5DGo). Under Adx conditions SPC6 labeled corticotrophs increased nearly 5-fold (from 12% to 60%). The greatest increase in mRNA levels was observed for SPC4 (Fig. 5CGo). Under Adx conditions SPC4 increased nearly 2-fold. In the case of SPC3 (Fig. 5BGo) significant changes were observed after Adx treatment, both in the percentage of labeled cells and in mRNA levels. It was also noted that Dex treatment reversed each of the changes observed after Adx treatment for SPC1, SPC3, SPC4, and SPC6. As a final note, there were no observed changes in SPC expression after ADX and Dex in the neurointermediate lobe.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Some of the most important current issues regarding the biological function of the family of mammalian SPCs are in regard to their substrate specificity and potential overlapping activities (6). Various studies are currently investigating SPC structure as well as cleavage specificity in cellular overexpression systems (21, 22, 23) and in vitro assays (24, 25, 26). However, an understanding of the precise localization of these enzymes in vivo is also important to determine whether overlapping cleavage specificity observed in vitro could have any impact within the physiological content. The first step to this understanding is to define the exact cellular localization of each enzyme. This is a complex task in heterogeneous tissues such as the pituitary. However, our dual labeling in situ hybridization methodology has the advantage of defining cellular localization while maintaining the integrity of the pituitary tissue (18). Furthermore, a specific cell type can be examined within the context of a regulatory paradigm such as that of manipulating corticosteroids (12). In previous work we have shown the gene expression of SPC1 (8), SPC2, SPC3 (9), SPC4, and SPC6 (10) in the pituitary. In present work, we examined the colocalization of the mRNAs of these five convertases with POMC, GH, PRL, TSH, and FSH mRNAs to identify the distribution pattern in individual cellular populations in the anterior pituitary. Interestingly, no individual pituitary cell population expressed a single convertase, but at least three SPC mRNAs were abundantly expressed in each cell type. Furthermore, we did not always detect SPC expression in all cells (>90%) of a defined population examined. A striking example was demonstrated for lactotrophs, which were observed to express SPC1 and SPC4 in nearly all cells examined, whereas only 50–55% of lactotrophs expressed SPC2, SPC3, and SPC6. This could be due to a technical difficulty, where the in situ methodology could not detect very low levels of mRNA expression. However, a more likely alternative explanation is that subpopulations of individual pituitary cell types are regulating the expression of the same SPC in a differential manner. The differential expression of SPCs within lactotrophs suggests a different biological output of peptides/hormones from at least two lactotroph subpopulations.

A particular focus of our study was the POMC-expressing corticotroph cell population. Previous studies have demonstrated the differential cleavage specificity of SPC2 and SPC3 in the processing of the POMC (22, 23), showing that ß- endorphin and {alpha}MSH are generated by SPC2 and that ACTH and ß-lipotropin are generated by SPC3. The general pattern of SPC2 and SPC3 expression fits with this processing specificity, as SPC2 is mainly expressed in the homogeneous intermediate lobe, but is not very highly expressed in the anterior lobe. In contrast, SPC3 is highly expressed in the anterior lobe. In the present study our data revealed a more complex situation within the corticotroph cell population, showing that a majority of corticotrophs (>80%) expressed SPC1, SPC3, and SPC4 mRNAs, whereas a minority of corticotrophs (<20%) expressed SPC2 and SPC6. It was therefore possible to conclude that convertases other than SPC3 could be involved in the anterior lobe processing of POMC. A role for SPC2 in corticotroph POMC processing was considered the most likely, because SPC2 has clearly been shown to cleave POMC using a variety of assay systems, including in vitro analysis (27), cellular overexpression systems (22, 23, 28, 29, 30), and knockout animals (31). The known anterior lobe processing pattern for POMC includes the presence of ß- endorphin, along with ß-lipotrophin, usually in approximately a 1:2 ratio (32). The anterior lobe also produces minor amounts of {alpha}MSH-like material (33). The presence of SPC2 in a subpopulation of corticotrophs is therefore a very likely explanation for the presence of low levels of ß-endorphin and {alpha}MSH-like peptides in the anterior lobe. Alternative explanations have been proposed, including the secondary cleavage specificity of SPC3 for the ß-endorphin site (22). However, only minute amounts of ß-endorphin were generated using highly overexpressed SPC3, suggesting that SPC3 is very ineffective in generating ß-endorphin. Other SPCs expressed in corticotrophs could be potentially involved in POMC processing. However, the role of SPC1 is thought to be limited, because it cleaves precursors that enter the constitutive secretory pathway (34), and thus its differential intracellular localization may well preclude a physiological role in POMC processing. Similarly, recent studies also seem to exclude SPC4 from a role in POMC processing due its very distinct intracellular trafficking (35). Although the exact intracellular compartment of SPC4 has not been identified, coexpression studies with POMC in corticotroph-derived cell lines do not show any effect of SPC4 on POMC processing (35). As for a potential role of SPC6 in POMC processing, any conclusion would be premature, as the cleavage specificity of SPC6 on POMC has not been reported. However, the intracellular colocalization of SPC6 with POMC appears possible, because studies have shown that the soluble form of this enzyme (i.e. SPC6A) can be directed to secretory granules (36), and this form is also present in the anterior pituitary (37). The role of SPCs in corticotrophs is certainly not limited to POMC processing, as other peptides, such as gastrin-releasing peptide (1), galanin, and neuromedin U (2), have been localized in corticotrophs.

The hormonal and pharmacological regulation of SPC gene expression has been well documented. SPCs have been shown to be regulated by thyroid status (9, 38), dopaminergic agonists and antagonists (9), cAMP (39, 40, 41), TGFß (42), seizure activity (43), retinoic acid (44), and glucocorticoids (9, 12). Our studies have focused on SPC gene expression in the HPA axis. It is well known that glucocorticoids may exert their effects directly on the corticotrophs via the classical type II GR or through their negative feedback effects via the inhibition of hypothalamic CRH neurons. We have shown in hypothalamic parvocellular CRH neurons the induction of SPC3 mRNA after removal of endogenous glucocorticoids via adrenalectomy (12). These data suggested an active role of SPCs in the determination of the peptidergic output of hypothalamic neurons implicated in the stress response and ultimately in the regulation of pituitary corticotrophs. Using a similar paradigm, in the present study we examined the effects of corticosteroid status on SPCs expressed in the anterior lobe corticotrophs. Our present findings showed that SPC3 mRNA expression was up-regulated after adrenalectomy and that this effect was reversed with Dex treatment. Interestingly, SPC3 was not the only convertase to be up-regulated in corticotrophs. Similar results were obtained for SPC1 and SPC4. Furthermore, a dramatic change was observed for the corticotroph expression of SPC6, with an almost 2-fold increase in mRNA levels appearing in 5- to 6-fold more corticotrophs than under normal conditions. These results contrast with our previous observation of SPC3 mRNA induction in CRH hypothalamic neurons (12), as in those neurons we did not observe any changes in SPC1, SPC2, SPC4, or SPC6 with the removal of glucocorticoids. However, removal of glucocorticoids results in an increased drive from the hypothalamic neurons, which could stimulate corticotrophs through the actions of CRH and vasopressin. We also noted that treatment with Dex alone did not significantly reduce basal expression levels of SPCs in corticotrophs, although they clearly reversed the effects of Adx. These data suggest that corticosteroids are not regulating SPC expression directly at the pituitary level, but, rather, through actions at CRH hypothalamic neurons.

Although individual components of the HPA axis have been extensively examined, the molecular control mechanisms of the mammalian endocrine response to stress are not yet well defined. Furthermore, the functional relationship of many neuropeptides in the stress axis is not understood. Certainly the plasticity of the pituitary corticotroph and the ability to generate diversity through precursor processing could have a significant impact on the functional response. The results of the present study suggest that SPCs may be directly involved in the mammalian stress response and may be important in HPA axis homeostasis. Additional studies on the impact of stress and/or glucocorticoid levels on SPC protein expression and cleavage activity will further address the in vivo role of SPCs in HPA axis regulation.


   Acknowledgments
 
We thank Dr. R. A. Maurer (Oregon Health Sciences Center, Portland, OR) for his generous gift of the rat FSH ß-subunit cDNA, and Dr. J. Eberwine (University of Pennsylvania, Philadelphia, PA), who kindly provided the rat POMC cDNA. We also thank Ms. Xue Wen Yuan for excellent technical support.


   Footnotes
 
This work was supported by a grant from the Canadian Institutes of Health Research (to R.D.).

1 Scholar (Chercheur Boursier Senior) of the Fonds de la Recherche en Santé du Québec. Back

Abbreviations: Adx, Adrenalectomy; Dex, dexamethasone; Dig, digoxigenin; h, human; HPA, hypothalamic-pituitary-adrenal; m, mouse; nt, nucleotides; r, rat; SPC, subtilisin-like proprotein convertase.

Received June 8, 2001.

Accepted for publication September 10, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Houben H, Denef C 1991 Evidence for the presence of gastrin-releasing peptide immunoreactivity in rat anterior pituitary corticotrophs and lactotrophs, AtT 20 cells and GH3 cells. Failure to demonstrate participation in local control of hormone release. Endocrinology 128:3208–3218[Abstract]
  2. Cimini V, van Noorden S, Timson CM, Polak JM 1993 Modulation of galanin and neuromedin U-like immunoreactivity in the rat corticotropes after alteration of endocrine status. Cell Tissue Res 272:137–146[CrossRef][Medline]
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