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Endocrinology Vol. 142, No. 9 4141-4149
Copyright © 2001 by The Endocrine Society

ARTICLES

Coexpression of Proprotein Convertase SPC3 and the Neuroendocrine Precursor ProSAAS1

Edith Lanoue and Robert Day

Department of Pharmacology, 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., Département de Pharmacologie, Faculté de Médecine, Institut de Pharmacologie, 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 are a family of serine proteinases involved in the processing of secreted proteins via cleavage at paired basic residues. Until recently, only one natural inhibitor had been demonstrated, the neuropeptide 7B2, which contains a C-terminal domain with inhibitory activity against SPC2. A novel granin-like peptide precursor, named proSAAS, has recently been identified that contains potent and specific inhibitory activity on SPC3 in vitro. To exert such an inhibitory action of SPC3 activity, it would be important to demonstrate that proSAAS and SPC3 are colocalized. We have studied the expression of proSAAS and SPC3 mRNAs in the rat central nervous system and various peripheral tissues by in situ hybridization histochemistry. Our results show that, like 7B2, proSAAS is expressed with a panneuronal distribution. In the periphery, proSAAS is an excellent marker of endocrine cells. Double labeling studies show that SPC3 expression is nearly always accompanied by proSAAS expression. However, proSAAS was also found to be expressed in endocrine cells and neurons that did not express SPC3, suggesting that proSAAS could have additional functions other than the modulation of SPC3 activity. These data support the hypothesis that one of the roles of proSAAS may be to modulate the activity of SPC3.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROTEIN PRECURSORS SYNTHESIZED within the secretory pathway undergo a number of posttranslational modifications before achieving full biological activity. Proteolytic processing at single and paired basic residues is recognized as an essential step to yield active hormones and neuropeptides. The consensus motif Lys/Arg-Xn-Arg (n = 0, 2, 4, or 6) is thought to be a primary site where proteolytic cleavage occurs, C-terminal to the P1 Arg (1, 2). The subtilisin-like proprotein convertases (SPCs) are now known to carry out this critical function. This family of enzymes consists of seven distinct members named SPC1 (furin/PACE), SPC2 (PC2), SPC3 (PC1/PC3), SPC4 (PACE4), SPC5 (PC4), SPC6 (PC5/PC6), and SPC7 (LPC/PC7/PC8) (3, 4, 5, 6). Each convertase has been shown to cleave precursors in vivo and in vitro at the recognized consensus motif of Lys/Arg-Xn-Arg (3, 4, 5, 6, 7, 8, 9, 10, 11, 12). Subsequent to SPC cleavage, the C-terminal basic residue must be removed via the action of carboxypeptidases such as carboxypeptidase E (CPE) (13, 14) or carboxypeptidase D (15, 16) before the peptides become bioactive. In some cases, additional posttranslational modifications occur, such as amidation, phosphorylation, or glycosylation (17, 18).

The distribution pattern of each SPC has been extensively investigated (3, 19, 20, 21). One insight obtained from these studies is that although distinct expression patterns are observed for each SPC, it is clear that all cells express at least two or more SPCs (3), suggesting coordinated or distinct functions within the intracellular environment. SPC1 has been shown to be expressed ubiquitously (19); in contrast, SPC2 and SPC3 have an expression pattern that closely follows that of endocrine and neural cells (20, 22). This is consistent with the notion that SPC2 and SPC3 process precursors that enter the regulated secretory pathway, mainly hormones and neuropeptides (4). The regulated secretory pathway has distinctive features that result in special requirements for SPC functioning within this pathway (23). Some of these are well studied, such as pH optimum, aggregation and condensation, and sorting to secretory granules. Among these special requirements is the ability to regulate enzyme activity in a temporal manner. This is the case for SPC2, whose activation process requires a partner protein, known as 7B2 (22, 24, 25, 26, 27). 7B2 is a bifunctional protein that contains an N-terminal domain (21 kDa) that is critical for the activation of SPC2. However, 7B2 also contains a C-terminal domain (C-T peptide) that has potent (i.e. nanomolar) but transient inhibitory activity on SPC2 (25, 28, 29). In vivo, the action of the C-T peptide is to delay SPC2 activation until the enzyme reaches the late Golgi compartments. C-T peptide inhibition of SPC2 is removed via its cleavage by SPC2 and the subsequent action of carboxypeptidases (30). Comprehensive distribution studies of 7B2 and SPC2 showed that they are always coexpressed (22), strongly supporting the biochemical and cellular function studies. However, 7B2 was also shown to have a much larger distribution than SPC2, both in the central nervous system (CNS) and in the periphery. In the CNS, 7B2 had a panneuronal distribution, whereas in the periphery, it was exclusively localized in endocrine cells (22). The widespread neuroendocrine distribution of 7B2 suggests that it is an excellent marker of neuroendocrine cells and further that it must have other functions than the exclusive regulation of SPC2 activity.

Until last year, 7B2 was the only known natural inhibitor for any SPC. Recently, a novel granin-like precursor, proSAAS, has been characterized (31). The discovery of proSAAS was made using Cpefat/Cpefat mice, which have defective processing due to lack of active CPE enzyme. The result is that these mice accumulate peptides with extended basic residues at their C termini. These peptides are generally associated with the regulated secretory pathway, because CPE is directed and functions within the regulated pathway. A number of peptides were isolated from Cpefat/Cpefat mice using a strategy to isolate peptides with basic residues in their C termini. Five of these peptides were recognized to be present within a single precursor, which was named proSAAS. It was then suggested that proSAAS could be an endogenous inhibitor of SPC3 based on its loose similarity to 7B2. Subsequent studies demonstrated a potent and specific inhibition of proSAAS peptides for SPC3 (31, 32, 33). The inhibitory sequence was localized to a C-terminal region that includes the sequence LLRVKR. These studies make a strong case for the possibility that proSAAS is a natural inhibitor for SPC3 in a manner analogous to that of 7B2 with SPC2. However, to strengthen the case for proSAAS function, studies regarding its expression patterns are justified, especially in comparison with 7B2 and SPC3. In the present study, we investigated the potential biosynthetic sites of proSAAS in endocrine and nonendocrine cell lines, in the CNS, and in various peripheral endocrine tissues. We specifically examined the codistribution patterns of SPC3 with proSAAS mRNA. Our data support the notion that one of the functions of proSAAS is to serve as an intracellular regulator of SPC3 activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
cRNA probe synthesis
All experiments carried out in these studies used antisense cRNA probes and sense probes for controls. Probes were synthesized by in vitro transcription of linearized plasmid DNA, and labeling was done by incorporation of radioactive ([35S]UTP/[35S]CTP, [33P]UTP, [32P]UTP) (Amersham Pharmacia Biotech, Arlington Heights, IL) and nonradioactive (digoxigenin-11-UTP) (Roche Molecular Biochemicals, Basel, Switzerland) nucleotides (34).

Rat proSAAS was cloned in pSPORT1 vector (Life Technologies, Inc., Gaithersburg, MD), and the transcribed cRNA probe consisted of 950 nucleotides corresponding to the full length coding region of proSAAS (31). For in situ hybridization of proSAAS, sense and antisense RNA probes were used in a nonradioactive form using digoxigenin-UTP incorporation and in a radioactive form using [33P]UTP labeling. Because the proSAAS probes were 950 nucleotides in length when used for in situ hybridization, a hydrolysis procedure was applied as described previously (34). Briefly, this method consists of incubating the probe in carbonate buffer (0.4 M NaHCO3/0.6 M Na2CO3) at 65 C for 15 min followed by neutralization and an ethanol precipitation step (0.03 M sodium acetate, pH 5.2, 0.1% acetic acid, 0.02 mg/ml yeast tRNA, 0.2 M ammonium acetate, and ethanol).

The antisense and sense [35S]UTP/[35S]CTP and [33P]UTP-labeled cRNA probes of rat SPC3 consist of the 590 nucleotides equivalent to segment 1841 to 2430 in mouse SPC3 (35). For 7B2, we used a cRNA [32P]UTP-labeled antisense probe consisting of the 1.1-kb full-length mouse 7B2 (36).

Northern blot analysis
Total RNA was extracted from cell lines using a guanidinium isothiocyanate methodology, which includes a lithium chloride precipitation step (19). The RNA (5 µg) was separated on an agarose gel containing 6% formaldehyde and blotted on Nytran Plus membranes (Schleicher & Schuell, Inc., Keene, NH). Prehybridization was carried out for 2 h at 65 C in hybridization buffer containing 5% SDS, 0.4 M sodium phosphate buffer, 1 mM EDTA, 1 mg/ml BSA, and 50% formamide. The purified [32P]UTP-labeled cRNA probe was then added to the prehybridization buffer and incubated with the Nytran membrane overnight at 65 C. Filters were washed in 0.1x sodium citrate, 0.1% SDS, 1 mM EDTA at 72 C for 2 h and exposed to x-ray film (XAR-5 film with intensifying screens; Eastman Kodak Co., Rochester, NY) at -80 C for 3 h.

In situ hybridization
The in situ hybridization studies were carried out using antisense and sense labeled cRNA probes in the following combinations: 1) for single label studies, [33P]UTP-labeled cRNA probe alone (proSAAS or SPC3); and 2) for colocalization studies, mixtures of [35S]UTP/[35S]CTP-labeled and digoxigenin-UTP-labeled cRNA probes. Radioactive probes were diluted to 33 x 103 dpm/µl, and in situ hybridization was performed as described previously (34). Sprague Dawley rats were killed by rapid decapitation, and tissues were rapidly removed and frozen in isopentane cooled to -35 C. The extracted tissues were stored at -80 C until cryosectioning. Frozen 10-µm sections were cut on a Reichert cryostat (Leica Corp., Depew, NY) and thaw-mounted on polylysine-coated glass slides and stored at -80 C until processing.

For the single label studies, sections were hybridized with proSAAS or SPC3 [33P]UTP-labeled cRNA probes. These sections were submitted to standard in situ hybridization procedures (34) followed by x-ray autoradiography for 1–7 d, resulting in low resolution images. These slides were also dipped in nitroblue tetrazolium nuclear emulsion (Eastman Kodak Co.) to obtain cellular resolution. The sections hybridized were counterstained with cresyl violet, cleared in xylene, and mounted with Permount histological mounting medium (Fisher Scientific, Pittsburgh, PA).

For the cellular colocalization studies, the sections were hybridized simultaneously with digoxigenin-UTP-labeled proSAAS and [35S]UTP/[35S]CTP-labeled SPC3 cRNA probes. Probes were applied to the sections in hybridization buffer (75% formamide, 10% dextran sulfate, 3x SSC, 50 mM sodium phosphate, pH 7.4, 1x Denhardt’s solution, and 0.1 mg/ml yeast tRNA) for 16 h at 55 C. After RNase A treatment and high stringency washes, digoxigenin-labeled probes were detected with an antidigoxigenin antibody conjugated to alkaline phosphatase. For detection of the radioactive probe, the slides were then dipped in Ilford K-5D emulsion (Polysciences, Warrington, PA). All emulsion-dipped slides were stored for 3 d to 10 wk at 4 C. Autoradiograms were developed in Kodak D-19 for 4 min and fixed in 30% sodium thiosulfate for 4 min. Sections hybridized with both radioactive and digoxigenin-labeled cRNA probes were mounted with Aquaperm mounting medium (Immunon, Pittsburgh, PA). As a negative control, radioactively labeled sense strand probes of the same size and specific activity were used instead of the antisense strand probe.

Cell lines
In these studies, we used a number of cell lines for comparative purposes. The following cell lines can be generally considered as of endocrine or nonendocrine origin. Neuro2A (mouse neuroblastoma), NG108 (rat-mouse neuroblastoma-glioma hybrid), SKNM (human neuroepithelioma), GH4C1 (rat pituitary somatomammotroph), GH3 (rat pituitary somatomammotroph), AtT-20 (mouse pituitary corticotroph), Rin m5F (rat insulinoma), BSC-40 (African green monkey kidney epithelial cells), PC12 (rat pheochromocytoma), ßTC-3 (mouse insulinoma), Y-1 (mouse adrenal cortex), NIH 3T3 (mouse embryo), Ltk- (mouse connective tissue), TM4 (mouse testicular Sertoli cells), and Caco2 (human colon adenocarcinoma).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Comparative Northern analysis of proSAAS and 7B2 in cell lines
Previous studies have examined the expression of 7B2 in various cell lines (37) and tissues (22, 37). Because proSAAS has been suggested to be related to the granin family of proteins (31), we decided to first explore the comparative expression of proSAAS with 7B2 in a number of endocrine- and nonendocrine-derived cell lines (Fig. 1Go). As previously shown (37), 7B2 was expressed exclusively in endocrine-derived cell lines such as Neuro-2A, NG108, GH4C1, GH3, AtT-20, Rin m5f, PC12, and ßTC3. The same blot was used for hybridization with a proSAAS probe (after completely stripping the 7B2 signal). The results show identical expression patterns of proSAAS and 7B2. The 7B2 band was observed at about 1.2 kb, whereas that of proSAAS was slightly lower at about 1.0 to 1.1 kb.



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  		Figure 1. Northern blot analysis of cell lines for 7B2 (top) and proSAAS (bottom) mRNA. In each lane, 5 µg of total RNA was loaded and analyzed as described in Materials and Methods. For detection of mRNA, the x-ray film was exposed for 3 h. Molecular mass markers are shown on the right.

 
Comparative distributions of proSAAS and SPC3 mRNAs in the rat CNS
Our previous studies examined the expression of 7B2 in the rat CNS, with a close comparison with SPC2 (22). In a similar manner, we examined the expression pattern of proSAAS with that of SPC3 (Figs. 2Go and 3Go). These broad in situ hybridization studies were carried out on consecutive sections throughout various brain regions. It was first noted that the distribution of proSAAS is very widespread in a manner analogous to that of 7B2 (22). In contrast, SPC3 distribution is more restricted than that of proSAAS. Thus, just as SPC2 has a more restricted distribution compared with 7B2 (22), so does SPC3 compared with proSAAS (Figs. 2Go and 3Go). At this low level of resolution, we notice that there is a high overlap of expression of proSAAS and SPC3, for example, within the supraoptic and paraventricular nuclei (Fig. 3Go, C and D) and the hippocampus (Fig. 3Go, G and H). Although an extensive comparative quantitative analysis was not performed, we believe that proSAAS mRNA levels are most likely higher in most regions that express SPC3, because the x-ray film exposure time was much shorter (1 day for proSAAS compared with 6 days for SPC3). Once again, these results are strikingly similar to those obtained previously in the comparison of 7B2 and SPC2 mRNA expression in the rat brain. Control sections were hybridized with sense proSAAS or sense SPC3 cRNA probes, but no signal was observed.



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  		Figure 2. Distribution of proSAAS (A) and SPC3 (B) in the sagittal section of rat brain. SPC3 expression appears to overlap with that of proSAAS. CBL, Cerebellum; DG, dentate gyrus; Pir, piriform cortex. Bar, 6 mm.

 


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  		Figure 3. Consecutive sections showing autoradiographic images of proSAAS (A, C, E, G, and I) and SPC3 (B, D, F, H, and J) mRNA distributions in rat brain coronal sections. SPC3 expression patterns appear to always be overlapped by proSAAS expression. No positive labeling was observed in control sections labeled with proSAAS and SPC3 sense probe (K and L). AD, Anterodorsal thalamic nucleus; CBL, cerebellum; CTX, cerebral cortex; DG, dentate gyrus; Hpc, hippocampus; IC, inferior colliculus; MHb, medial habenular nucleus; MVe, medial vestibular nucleus; Pir, piriform cortex; PVN, paraventricular hypothalamic nucleus; PVA, paraventricular thalamic nucleus, SHi, septohippocampal nucleus; SNC, substantia nigra; SO, supraoptic nucleus. Bar, 4 mm.

 
We then examined the comparative distribution of proSAAS and SPC3 mRNAs at a cellular resolution, but still on consecutive sections. Thus, the same sections shown in Fig. 3Go were emulsion dipped for these studies, and some representative examples are shown in Fig. 4Go. These studies demonstrated that both proSAAS and SPC3 signals could only be observed over neurons. We can identify neuronal elements by using a cresyl violet counterstain that we have validated previously for the in situ hybridization procedure (34). As shown in Fig. 4Go, a very strong correlation between proSAAS and SPC3 is observed in the labeling of paraventricular nuclei (Fig. 4Go, A and B) and the hippocampus (Fig. 4Go, C and D). These results suggest that SPC3 mRNA is coexpressed with proSAAS.



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  		Figure 4. Adjacent sections showing proSAAS (A and C) and SPC3 (B and D) expression in the paraventricular nucleus (PVN) and hippocampus (Hpc). Tissue sections were emulsion dipped to obtain cellular resolution images. Arrows indicate labeled neurons. Neurons were identified using a cresyl violet counterstain. Bar, 100 µm.

 
Colocalization of SPC3 with proSAAS
To obtain more direct information concerning the expression of proSAAS and SPC3, we carried out double labeling experiments on the same tissue sections using a nonradioactively labeled proSAAS cRNA probe and a radioactively labeled SPC3 cRNA probe. Figure 5Go shows examples of the colocalization of proSAAS and SPC3 mRNAs in the Purkinje cell layer (Fig. 5AGo) and in the supraoptic nucleus (Fig. 5BGo). From these studies, we also determined that SPC3 mRNA expression in the brain was always associated with proSAAS. However, we could also identify many cells in which proSAAS mRNA expression was very strong without any evidence of SPC3 signal, such as in the pontine nucleus (Fig. 5CGo).



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  		Figure 5. Colocalization study of proSAAS and SPC3 mRNA in rat brain within the Purkinje cell layer (PCL) (A), the supraoptic nucleus (SON) (B), and the pontine nucleus (Pn) (C). D shows a control, using a sense strand digoxigenin-labeled proSAAS probe. A and B show examples of proSAAS and SPC3 when they are colocalized. ProSAAS mRNA was detected using a nonradioactive labeled probe resulting in the purple-brown color, whereas SPC3 was detected using a radioactively labeled probe resulting in the silver grains observed. The arrows point to cells that show the colocalization of proSAAS and SPC3. C shows an example of neurons that express proSAAS but not SPC3. Arrowheads point to neurons that show intense purple-brown color but do not shown any silver grains. Bars, 25 µm for A, B, and C, 100 µm for D.

 
ProSAAS and SPC3 expression in peripheral tissues
Because proSAAS and 7B2 have remarkably similar expression patterns in the brain, it was of interest to determine if this correlation could also be substantiated in the peripheral tissues within endocrine cells. Furthermore, it would be of interest to determine if proSAAS and SPC3 expression were also colocalized in these same tissues. Using in situ hybridization, we observed proSAAS expression in the pituitary (Fig. 6AGo) and adrenal medulla (Fig. 6CGo). In the pituitary, proSAAS was very highly expressed in both the intermediate and anterior lobes. As demonstrated previously (19), SPC3 pituitary expression was also observed in both anterior and intermediate lobes. The heterogeneous nature of the anterior lobe prohibits interpretations of the colocalization of proSAAS and SPC3. However, in light of the homogeneous nature of the intermediate lobe, we can state that proSAAS and SPC3 are colocalized within melanotrophs. At the level of the adrenal, both proSAAS and SPC3 mRNAs were observed within the adrenal medulla, which consists of chromaffin cells. It is interesting that proSAAS signal could also be observed in the outer edge of the cortex, which is the glomerulous cell layer responsible for the biosynthesis of aldosterone. We also examined the expression of proSAAS in the testis and observed expression within the interstitial cells (Fig. 6Go, E and F), possibly within Leydig cells. No SPC3 signal could be observed in the testis. Finally, the codistribution of proSAAS and SPC3 mRNAs was examined within the small intestine (i.e. ileum). Low power magnification (Fig. 7AGo) of proSAAS expression reveals a very strong, yet localized, signal in the outer edges of the intestinal sections. In contrast, the SPC3 signal (Fig. 7BGo) was more discrete and more restricted to the internal layers. High power magnification shows that proSAAS was in fact localized to ganglionic cells in the external muscular layer, whereas SPC3 was presumably localized with endocrine cells embedded within the intestinal epithelium, as described previously (38, 39). The SPC3-positive cells did not display proSAAS expression.



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  		Figure 6. Distribution of proSAAS and SPC3 mRNA in consecutive rat tissue sections, including the pituitary (A and B), the adrenal (C and D), and the testis (E and F). In the pituitary, both mRNAs were observed in the anterior (AL) and intermediate lobes (IL). ProSAAS and SPC3 labeling was observed in the adrenal medulla (AM); however, proSAAS mRNA was also observed in the glomerulosa (C). In E, a dark-field x-ray image of proSAAS mRNA shows an extensive distribution pattern, whereas in F, the emulsion-dipped section reveals labeling of proSAAS in the interstitial cells, most likely within Leydig cells (arrows). Bars, 3 mm for A through E; 300 µm for F.

 


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  		Figure 7. ProSAAS (A and C) and SPC3 (B and D) mRNA expression in rat intestine. A and B are low power autoradiographic x-ray images showing the intense labeling of proSAAS and SPC3 in distinct areas. In C and D, emulsion-dipped sections examined at the light microscope level reveal that proSAAS is expressed in the outer muscle layer in ganglionic cells (C, arrows), whereas SPC3 is expressed within the intestinal epithelium (D, arrows), most likely within endocrine cells. Bars, 5 mm for A and B, 100 µm for C and D.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The granin family of precursors of proteins is a loosely defined group that includes chromogranin A, chromogranin B, secretogranin II, 7B2 (40, 41), and possibly proSAAS. Although a common feature of this family of proteins is their strict pattern of expression localized mainly to neural and endocrine tissues and cells, their functions are not well defined. Some studies have suggested that chromogranin A, chromogranin B, and secretogranin II may be precursors of bioactive peptides (42), but they could also be implicated in the sorting of proteins into the regulated secretory pathway (41, 43). The case of 7B2 appears to be better defined because there is strong evidence to show that it has dual function as an inhibitor and an activator of SPC2 (24, 25, 26). The novel granin-like precursor proSAAS appears to have some functional similarities to 7B2, because its inhibitory activity against the convertase SPC3 has been reported. This parallel function within the regulated secretory pathway led us to investigate the distribution of proSAAS in relation to SPC3 in the CNS and various endocrine tissues.

Our present study shows that, like 7B2 (22), proSAAS has a broad distribution in the CNS that is exclusive to neurons and can be considered as panneuronal. Furthermore, the expression of proSAAS was only observed in endocrine-derived cell lines, which also express 7B2. This very high correlation of expression in the CNS and in cell lines suggests that the expression of proSAAS is strongly linked to the presence of a regulated secretory pathway in a manner akin to that of 7B2. Thus, even though there is no amino acid sequence homology, the close correlation of expression of proSAAS within neuroendocrine cells allows it to be categorized as a granin-like precursor.

With regard to the function of proSAAS, previous studies demonstrated the inhibitory activity of proSAAS on SPC3 (31, 32, 33). The inhibitory region has been localized to a sequence in the C-terminal region of the precursor (i.e. LLRVKR) (31, 32). This peptide has been shown to have highly potent (i.e. nanomolar) and specific inhibition on SPC3. Support for this inhibitory function in vivo requires the demonstration of proSAAS and SPC3 colocalization. Our studies show that in the CNS, SPC3 neurons always coexpress proSAAS (Fig. 5Go). Similarly, in peripheral endocrine tissues, the intermediate pituitary, and the adrenal medulla, SPC3 and proSAAS expression were highly correlated. The colocalization results of proSAAS with SPC3 are similar to those demonstrated previously for 7B2 with SPC2 (22). Like 7B2 in relation to SPC2, proSAAS had a much more extensive distribution than SPC3. In both studies, the expression of proSAAS and 7B2 was much higher than that of SPC3 and SPC2, respectively. These data suggest that both proSAAS and 7B2 may have other functions than those associated with SPC3 and SPC2. Because proSAAS is extensively processed (44), it may also be a "true" neuropeptide precursor. However, this function awaits the demonstration of receptors and signaling functions for proSAAS-derived peptides.

The inhibitory function of proSAAS is highly dependent on how this precursor is processed in vivo. Cleavage at the C terminus of the proSAAS peptide (i.e. LLRVKR{downarrow}) would result in the exposure of the inhibitory site to carboxypeptidase enzymes, namely CPE, within the regulated secretory pathway. Removal of these C-terminal basic residues would result in the removal of the inhibitory potency of the proSAAS peptide for SPC3. A recent study examining the processing of the proSAAS precursor has shown that the proSAAS peptide is cleaved at the inhibitory site (44). Thus, a fully processed proSAAS peptide (lacking the C-terminal Lys-Arg residues) is accumulated in neuroendocrine cells. These data suggest that the proSAAS peptide can only inhibit SPC3 in a transient way, within the intracellular environment. Larger forms of the proSAAS peptide (such as the PEN/LEN peptide) would protect the inhibitory cleavage site from the action of CPE; however, these peptides are not accumulated in normal mice (44). This contrasts with the situation of pro7B2 and its inhibitory peptide known as the 7B2 C-T peptide. The intact 7B2 C-T peptide is accumulated in neuroendocrine cells and can thus inhibit SPC2 activity (30). Cleavage in the mid portion of this peptide by SPC2 or by other enzymes results in the exposure of the Lys-Lys inhibitory site, which can then be removed by CPE to terminate its action on SPC2. The fact that the PEN/LEN peptide does not accumulate like the 7B2 C-T peptide suggests a different temporal mechanism of inhibition.

Although our data strongly suggest that proSAAS is an excellent marker of neuroendocrine cells, we have observed some "atypical" localization that suggests some divergence from this concept. In the gastrointestinal system, specifically in the ileum, we observed a very high expression of proSAAS in ganglionic cells of the outer muscular layer. However, within the internal layers, where SPC3 is known to be localized within endocrine cells (38, 39), proSAAS expression could not be detected. Should the proSAAS peptide be important in the temporal inhibition of SPC3, this would not occur in these endocrine cells. It is not clear what effect the lack of proSAAS has on SPC3 activity in these endocrine cells. We have also observed a significant expression of proSAAS in the testis, specifically within interstitial cells, possibly including Leydig cells. The function of proSAAS at this level would only be speculative at this point. However, Leydig cells have been shown to have the capacity to express prodynorphin (45), proenkephalin (46), POMC (47, 48), and oxytocin (49). Finally, we have also observed an atypical expression of proSAAS mRNA in the adrenal cortex, in the glomerulosa, the outer cell layer that is responsible for the production of aldosterone. SPC3 is not expressed in the glomerulosa, and thus the presumed function of proSAAS at this level is also unknown.

In conclusion, the present study demonstrates that proSAAS is a good marker of neuroendocrine cells and that SPC3 and proSAAS expression is highly correlated. Although the association of proSAAS and SPC3 is highly similar to that of 7B2 and SPC2, distinct mechanisms of intracellular inhibition are highly likely.


   Acknowledgments
 
We thank Ms. Xue Wen Yuan for excellent technical assistance and Dr. Martin K.-H. Schafer (University of Marburg, Germany) for helpful discussions. We also thank Dr. Lloyd D. Fricker for providing us the proSAAS cDNA.


   Footnotes
 
1 This work was supported by grants from the Canadian Institutes of Health Research. R.D. is a scholar of the Fonds de la Recherche en Santé du Québec. Back

Abbreviations: CNS, Central nervous system; CPE, carboxypeptidase E; C-T peptide, C-terminal domain; SPC, subtilisin-like proprotein convertase.

Received March 13, 2001.

Accepted for publication May 24, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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