Differential Expression and Processing of Chromogranin A and Secretogranin II in Relation to the Secretory Status of Endocrine Cel
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《内分泌学杂志》
Department of Cell Biology, Physiology
Immunology, University of Cordoba (J.R.P., R.V.-M., D.C.G., A.R.-N., F.G.-N., J.P.C., M.M.M.), 14014 Cordoba, Spain
Institut National de la Sante et de la Recherche Medicale, Unite 413, European Institute for Peptide Research, Laboratory of Cellular and Molecular Neuroendocrinology, Unite Associee Centre National de la Recherche Scientifique, University of Rouen (Y.A., M.C.T., H.V.), 76821 Mont Saint Aignan, France
Abstract
Chromogranin A (CgA) and secretogranin II (SgII) are neuroendocrine secretory proteins that participate in regulation of the secretory pathway and also serve as precursors of biologically active peptides. To investigate whether there is a relationship between the expression, distribution, and processing of CgA and SgII and the degree of secretory activity, we employed two melanotrope subpopulations of the pituitary intermediate lobe that exhibit opposite secretory phenotypes. Thus, although one of the melanotrope subtypes shows high secretory activity, the other exhibits characteristics of a hormone storage phenotype. Our data show that SgII expression levels were higher in secretory melanotropes, whereas CgA expression showed similar rates in both cell subsets. The use of various antibodies revealed the presence of the unprocessed proteins as well as three CgA-derived peptides (67, 45, and 30 kDa) and six SgII-derived peptides (81, 66, 55, 37, 32, and 30 kDa) in both subpopulations. However, the smallest molecular forms of both granins predominated in secretory melanotropes, whereas the largest SgII- and CgA-immunoreactive peptides were more abundant in storage melanotropes, which is suggestive of a more extensive processing of granins in the secretory subset. Confocal microscopy studies showed that CgA immunoreactivity was higher in storage cells, but SgII immunoreactivity was higher in secretory melanotropes. Taken together, our results indicate that SgII and CgA are differentially regulated in melanotrope subpopulations. Thus, SgII expression is strongly related to the secretory activity of melanotrope cells, whereas CgA expression may not be related to secretory rate, but, rather, to hormone storage in this endocrine cell type.
Introduction
CHROMOGRANIN A (CgA) and secretogranin II (SgII) are members of the chromogranin/secretogranin family of proteins, collectively referred to as granins. The granin family also includes chromogranin B (CgB; also known as SgI), secretogranin III (SgIII/1B 1075), secretogranin IV/HISL-19 antigen (SgIV), and 7B2 (also known as secretogranin V; SgV) (1, 2). More recently, three new proteins, BRCA1 (3), NESP55 (4), and pro-SAAS (5), have also been proposed to belong to this family. Typically, granins are highly acidic proteins with an exclusive expression in endocrine, neuroendocrine, and neuronal cells. They are major constituents of secretory granules, in which they are costored with hormones and other products of the regulated secretory pathway (1, 6). Granins have been proposed to participate in the sorting and packaging of hormones and peptide precursors into secretory granules (7), yet the precise role of each granin within the secretory pathway is not completely understood. In addition to their intracellular functions, granins contain multiple dibasic amino acid cleavage sites and are considered precursors of peptides with autocrine, paracrine, or endocrine actions (8).
CgA was the first member of this family to be identified (9), and despite being the most studied granin, its precise function in neurosecretion remains controversial. Thus, although some researchers propose that CgA acts as an on/off switch, controlling secretory granule formation (10), others suggest that it may participate as a simple cargo protein (11) or act as an assembly factor of newly synthesized proteins (12). As a prohormone, CgA is the precursor of several active peptides, including the 14-amino-acid peptide WE14, which modulates histamine secretion in mast cells (13). Pancreastatin, vasostatin I and II, catestatin, parastatin, and chromacin are other CgA-derived peptides with known biological activities (Refs. 14, 15, 16, 17, 18 ; reviewed in Ref.19). Another potential peptide contained in the CgA sequence is the C-terminal 35-amino-acid fragment EL35, which is present in rat pituitary and adrenal glands (20).
SgII has been proposed to act as an assembly factor or helper of granule formation based on its ability to generate granule-like structures when expressed in cell lines lacking a regulated secretory pathway (21). SgII is processed in different neuroendocrine tissues (22, 23, 24, 25, 26), although only the 33-amino-acid-derived peptide secretoneurin (SN) has been shown to have biological activity (reviewed in Ref.27). Other fragments contained in the SgII sequence that are flanked by dibasic sites are EM66, which corresponds to a highly conserved region of the protein (28), and the recently identified peptide manserin (29).
As is the case for other protein precursors, the extent of cleavage of granins and, thus, the number and type of derived peptides are tissue and species specific (20, 30, 31). Additionally, granin expression is regulated in a cell-specific manner in response to a variety of stimuli (reviewed in Ref.32). In this study we attempted to characterize the expression, processing pattern, and intracellular distribution of CgA and SgII in relation to secretory activity in normal, nontumoral endocrine cells. To this end, we used melanotropes of the frog pituitary intermediate lobe as a cellular model. Melanotropes synthesize the precursor protein proopiomelanocortin (POMC), which is proteolytically processed in these cells to -MSH (33). Detailed ultrastructural and functional analyses of melanotropes have revealed that these cells can acquire two opposite secretory phenotypes, high secretory and biosynthetic activity (i.e. active secretory phenotype) or, alternatively, hormone storage (i.e. hormone storage phenotype), that coexist in the gland as two distinct cell subtypes containing a low amount of secretory granules (secretory melanotropes) or a highly granulated cytoplasm (storage melanotropes), respectively (34, 35, 36, 37, 38, 39, 40). To be more specific, although secretory melanotropes show low intracellular -MSH content, but display high POMC mRNA levels and -MSH secretory rate, storage melanotropes exhibit high -MSH intracellular levels, but low POMC mRNA content and -MSH release (34, 35, 36, 37, 38, 39, 40). Armed with this powerful model, we explored the possible relationship between these opposite secretory phenotypes and several features of CgA and SgII. Interestingly, we observed that although both CgA and SgII are expressed and processed in both melanotrope subtypes, the degree of granin processing was strongly dependent on the secretory status of the cells. Moreover, the two granins are differentially regulated in secretory and storage melanotropes at both the mRNA and protein level, suggesting distinct intracellular roles for CgA and SgII in the functioning of the regulated secretory pathway.
Materials and Methods
Reagents
The following materials were obtained from the indicated sources: Percoll and the ECL Plus kit were purchased from Amersham Biosciences (Barcelona, Spain); RNA-easy Mini Kit columns and ribonuclease-free deoxyribonuclease from Qiagen (Hilden, Germany); SuperScript II, Taq-DNA polymerase, ribonuclease inhibitor, deoxy-NTP mixture, and goat antirabbit conjugated to Alexa 594 from Molecular Probes (Barcelona, Spain); the protease inhibitors chymostatin, leupeptin, antipain, and pepstatin A and 5-bromo-4-chloro-3-indolyl phosphate from Roche (Barcelona, Spain). All other reagents were purchased from Sigma-Aldrich Corp. (Madrid, Spain).
Animals
All animal manipulations were performed according to the recommendations of the local ethical committees and under the supervision of authorized investigators. Adult frogs (Rana ridibunda) of about 40 g body weight (Ranas Orense, Mosqueiro, Spain) were maintained at 8 C on a 12-h light, 12-h dark photoperiod for at least 1 wk before death. The animals were killed by decapitation, and neurointermediate lobes were carefully dissected under a microscope. Glands were transferred to sterile Leibovitz culture medium (L-15) diluted 2:3 to adjust to R. ridibunda osmolality and supplemented with 1 mM glucose and 0.4 mM CaCl2 (pH 7.4). For mRNA and protein extractions, samples were frozen in liquid nitrogen and stored at –80 C until use.
Cell dispersion and isolation of melanotrope cell subtypes
Neurointermediate lobes (n = 30–40/experiment) were enzymatically and mechanically dispersed as described previously (34) until a homogeneous cellular suspension was obtained. For separation of the two melanotrope subsets, dispersed cells (1–2 x 106 cells in 250 μl culture medium) were layered on a 9-ml hyperbolic density gradient (1.027–1.072 g/ml) of Percoll (34). After centrifugation of the gradient (3000 x g, 25 min, 4 C), nine fractions of 1 ml each were collected manually. Fractions 5–7 contained secretory melanotropes (low-density cells), and fraction 1 contained storage melanotropes (high-density cells). The proportions of melanotropes recovered from these fractions were comparable to those previously reported (34, 35).
RNA isolation
Total RNA from whole cell populations of frog neurointermediate lobe or from the separate melanotrope subsets was isolated using RNA-easy Mini Kit columns followed by ribonuclease-free deoxyribonuclease treatment. The concentration and purity of the RNA samples were determined by UV spectroscopy at 260/280 nm.
Semiquantitative RT-PCR
The expression levels of CgA and SgII mRNAs in the two melanotrope cell subtypes were measured by semiquantitative RT-PCR, using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a housekeeping gene. Primers for PCR amplification were designed with Oligo software (National Biosciences, Cascade, CO) based on the corresponding cDNA sequences from R. ridibunda as follows: GAPDH sense, 5'-TTTCACCGCTACACAGAAG-3'; and GAPDH antisense, 5'-GTTGCTGTAACCGAATTCA-3' for the amplification of a 426-bp fragment of GAPDH cDNA; CgA sense, 5'-CGAGGAGATGAAAGGATTA-3'; and CgA antisense, 5'-ATCCTCATCGAATTGTCCT-3' for the amplification of a 574-bp fragment of CgA cDNA; and SgII sense, 5'-AGGCCAAGCAAACAACAAG-3'; and SgII antisense, 5'-CACCAAACTTCCTGCCATC-3' for the amplification of a 502-bp fragment of SgII cDNA.
RT and PCR were run in two separate steps. To enable appropriate amplification in the exponential phase for each target gene, PCR amplifications of GAPDH, CgA, and SgII transcripts were carried out in separate reactions with a different number of cycles (see Results), using the corresponding cDNA templates generated in single RT reactions. Briefly, equal amounts of total RNA (0.5 μg) from frog neurointermediate lobes or from the separate melanotrope subsets were heat denatured and reverse transcribed by incubation at 42 C for 60 min with 200 U SuperScript II, 40 U ribonuclease inhibitor, 10 μM deoxy-NTP mixture, and 250 ng random hexamer primers in a final volume of 40 μl 1x SuperScript II buffer. The reactions were terminated by heating at 75 C for 5 min and cooling on ice. For the optimization of the semiquantitative RT-PCR procedure, different concentrations of cDNA obtained from dispersed neurointermediate lobes and different number of cycles were tested for each gene to ensure amplification in the exponential phase. The reactions were carried out in a final volume of 50 μl 1x PCR buffer in the presence of 5 U Taq-DNA polymerase, 10 μM deoxy-NTP mixture, and the appropriate primer pairs (10 μM of each primer). PCRs consisted of, firstly, a denaturing cycle at 95 C for 5 min, followed by the number of cycles selected for optimal amplification of each gene, 30 sec at 95 C for denaturation, annealing at 55 C, and an extension cycle at 72 C for 1 min. A final extension cycle at 72 C for 7 min was included. PCR products were quantified at the end of amplification by electrophoresis on agarose gel (1%) containing ethidium bromide and measurement of signal intensity with Quantity-One software (Bio-Rad Laboratories, Inc., Hercules, CA). mRNA levels of CgA and SgII in each sample were calculated as a ratio of that for GAPDH in the same sample. The specificity of the PCR procedure was checked by omission of the cDNA template in the amplification reaction.
Western blot analysis
The study by Western blotting of CgA and SgII proteins in melanotrope cell extracts was carried out according to the method of Laemmli (41) with the appropriate modifications. In brief, cells were resuspended in 0.01 M PBS (pH 7.4) containing 20 μg/ml of the protease inhibitors chymostatin, leupeptin, antipain, and pepstatin A; sonicated (30 sec); and centrifuged at 3000 rpm for 5 min at 4 C to remove undisrupted cells and debris. Before electrophoresis, cellular extracts (20 μg protein) were suspended in 5x Laemmli buffer [62.5 mM Tris, 12.5% glycerol, 1.25% sodium dodecyl sulfate, 2.5% -mercaptoethanol, and 0.0125% bromophenol blue (pH 7.5)]. After boiling for 5 min, samples were separated by SDS-PAGE (10% acrylamide) using the Protean II Cell System (Bio-Rad Laboratories, Inc.) and blotted onto nitrocellulose membranes. Blots were stained with Ponceau S for visualization of protein bands and to confirm equal protein loading for further comparative analysis. Then blots were destained, blocked during 2 h in blocking buffer [5% low-fat milk in Tris-buffered saline (pH 7.6) with 0.05% Tween 20], and incubated overnight at 4 C with the corresponding specific antibodies. Two distinct regional-specific polyclonal rabbit antisera to human CgA (20) and SgII were used (23). Specifically, the antibodies were directed against the 14-amino-acid CgA-derived peptide WE14 (CgA273–285) and the C-terminal 35-amino-acid peptide EL35 (CgA344–378) for the analysis of CgA, and against SN and SgII187–252 (EM66) for the determination of SgII. Blots were then incubated for 1 h with a goat antirabbit IgG secondary antibody coupled to alkaline phosphatase. Visualization of the antigen/antibody complex was carried out by immersion of the blots in developing buffer [100 mM Tris, 100 mM NaCl, and 5 mM MgCl2 (pH 9.0)] containing nitro blue tetrazolium salt (7.5%) and 5-bromo-4-chloro-3-indolyl phosphate (5%). After immunostaining for granins, blots were allowed to dry, and the intensities of the immunoreactive bands were measured (see below). Thereafter, blots were subjected to a second immunostaining sequence using anti--actin and a horseradish peroxidase-conjugated secondary antibody. Blots were then revealed by chemiluminescence with the ECL Plus kit according to the manufacturer’s instructions. Signal intensities of immunoreactive bands were evaluated with the Quantity-One software described above. Quantitative data from the immunoreactive bands revealed with the anti-CgA anti-SgII sera were normalized against the corresponding -actin values measured on the same blots.
As a control of the specificity of the immunoreaction, blots from samples run in parallel were incubated in blocking reagent alone under the same conditions as those incubated with each specific antiserum. All cell extracts gave negative results when the primary antibodies were omitted.
Confocal microscopy
After dispersion of frog neurointermediate lobes and separation of the melanotrope subpopulations in density gradients, cells were washed in PBS. Fifty-microliter aliquots containing 40,000 cells were plated on slides. Cells were fixed in 4% paraformaldehyde for 15 min, rinsed in PBS, and incubated in the same buffer containing 1% BSA and 0.3% Triton X-100 for 1 h. Thereafter, cells were incubated overnight at 4 C in a humid atmosphere with anti-EL35 or anti-WE14 (CgA) and anti-EM66 or anti-SN (SgII) antibodies diluted in PBS containing 0.3% Triton X-100 and 0.5% BSA at a final dilution of 1:1000. The slides were then rinsed in PBS and incubated for 2 h with secondary antibody conjugated to Alexa 594 at a 1:500 dilution. Finally, the sections were rinsed in PBS and mounted with PBS/glycerol (vol/vol). The preparations were examined under a TCS-SP2-AOBS confocal laser scanning microscope attached to a Leica DM IRE2 inverted epifluorescence microscope equipped with a DMRXA2 lamp (Leica Corp., Heidelberg, Germany). To study the specificity of the immunoreaction, the following controls were performed: 1) omission of the specific antiserum, and 2) incubation with nonimmune rabbit serum instead of the primary antibodies.
The intensity of fluorescence signals in single secretory or storage melanotropes was monitored using an epifluorescence microscope (Eclipse TE2000-E, Nikon, Tokyo, Japan) fitted with a Plan-Fluor x60 oil immersion objective. The slides were epiilluminated at 540 nm for 800 msec for the acquisition of images of single cells. Image acquisition was controlled using Metafluor PC software (Universal Imaging Corp., West Chester, PA), and fluorescence emissions were captured using a CCD camera (C-4880-80, Hamamatsu, Hamamatsu City, Japan) running in 1-bit mode. The images acquired were transferred to off-line storage for subsequent analysis. Regions of the same field devoid of cells were selected for monitoring of the background.
Statistical analysis
For mRNA determinations, three independent RT-PCR experiments using RNA from different extractions were carried out for each target gene. For Western blot studies, three or four independent experiments using protein samples from different extractions were employed for the analysis of each granin. Results are expressed as normalized ratios by using GAPDH (for PCR studies) or -actin (for Western blot analysis) as reference. Data are expressed in arbitrary units (mean ± SEM). All comparisons were made between samples electrophoresed on the same gel and, in the case of the proteins, also blotted onto the same membrane. For analysis of the immunocytochemical signal in single cells to the four different antibodies, at least 20 storage melanotropes or secretory melanotropes obtained from four separate experiments were used. Statistical analysis was carried out with a paired t test (for RT-PCR and Western blot data) or Mann-Whitney rank-sum test (for immunofluorescence measurement in single cells) using the software package Statistica (StatSoft, Inc., Tulsa, OK). Differences were considered significant at P < 0.05.
Results
Semiquantitative RT-PCR analysis of CgA and SgII mRNA expression
The expression levels of CgA and SgII in melanotropes were evaluated by semiquantitative RT-PCR, using GAPDH as an internal standard. The technique was optimized for both transcripts by determining the number of PCR cycles and the amount of cDNA necessary for a quantitative amplification (i.e. within the exponential phase of PCR). Specifically, results from three separate experiments showed that 27 and 30 cycles of PCR amplification, corresponding to the linear portion of the curves for CgA and SgII, respectively, and 1 μl cDNA (equivalent to 25 ng total RNA), provided accurate amplification of both transcripts in mRNA samples from frog neurointermediate lobe and were therefore chosen for subsequent PCR quantifications. It should also be noted that the expression level of GAPDH provides an appropriate endogenous control, because its mRNA levels did not vary between the melanotrope cell subtypes (Fig. 1). No amplification products were obtained when the cDNA template was omitted from the PCR (data not shown).
Transcripts for both CgA (Fig. 1A) and SgII (Fig. 1C) were detected in mRNA extracts from the separate melanotrope cell subpopulations. Quantification of the 574-bp band corresponding to CgA revealed no differences in CgA mRNA content between the two melanotrope subtypes (Fig. 1B). In contrast, secretory melanotropes contained higher SgII mRNA levels than storage melanotropes (Fig. 1D).
Characterization of CgA in melanotropes
The analysis of CgA in frog melanotropes was carried out using two different antibodies: anti-EL35 and anti-WE14, which recognize the C-terminal end of CgA and a fragment of 14 amino acids located in a more internal region of the protein, respectively (20). Results were expressed as normalized ratios using -actin as the reference protein (Fig. 2).
Use of the EL35 antiserum revealed a single band of 67 kDa, which was apparent in extracts from both storage and secretory melanotropes (Fig. 2A). However, densitometric quantification of the band obtained for each melanotrope cell subtype showed a significantly stronger immunoreaction in extracts from storage melanotropes than from secretory cells (Fig. 2B). Likewise, the antibody against WE14 also labeled a 67-kDa band (Fig. 2C), which was more abundant in the storage cell subset than in secretory melanotropes (Fig. 2D). In addition, the WE14 antiserum revealed the presence of two additional fragments (45 and 30 kDa), which were differentially distributed in the two melanotrope cell subsets (Fig. 2C). Thus, although the smallest molecular form was hardly detectable in storage melanotropes, the intermediate mass band of 45 kDa, which displayed the strongest immunoreaction among the three bands appearing in the melanotrope subsets, clearly predominated in storage cells over secretory melanotropes (Fig. 2D). Finally, a weak immunostained band of 94 kDa was sometimes detected in extracts from storage cells (Fig. 2C).
Characterization of SgII in melanotropes
The antibodies used for this study, anti-SN and anti-EM66, recognize adjacent regions in the SgII sequence separated by a dibasic cleavage site (23, 42). These antibodies detected both common and distinct immunoreactive bands in melanotropes (Figs. 3 and 4). Specifically, anti-SN revealed a band of high molecular mass (66 kDa) as well as three bands of lower molecular masses (37, 32, and 30 kDa) in extracts from secretory and storage melanotropes (Fig. 3A). However, their relative abundance differed between the two melanotrope cell subtypes (Fig. 3B). Thus, densitometric quantification of the immunoreactive bands obtained for each cell subset and subsequent normalization of these values using -actin as a reference revealed that although the 66-kDa fragment was the most prominent SgII-derived cleavage product in storage melanotropes, the smallest, 30-kDa peptide predominated in the highly active cell subset. Immunostaining of both 37- and 32-kDa bands was numerically higher in secretory melanotropes than in storage melanotropes, although these differences did not reach statistical significance.
In addition to the 66- and 32-kDa bands, the antibody against the EM66 sequence also detected the presence of three other fragments of 97, 81, and 55 kDa in extracts from the two melanotrope cell subtypes (Fig. 4A). Occasionally, a weak immunopositive band of 30 kDa was observed in secretory melanotrope extracts (data not shown). Densitometric quantification showed no differences between storage and secretory melanotropes in the relative amount of either the two higher molecular mass fragments or in the 55-kDa fragment (Fig. 4B). However, in accordance with that found for the anti-SN antiserum, the 66-kDa form predominated in the melanotropes displaying low synthetic and secretory activities (Fig. 4B). In contrast, secretory melanotropes contained higher levels of the smallest molecular form of 32 kDa (Fig. 4B).
Confocal microscopy
Cellular distribution of CgA and SgII (Fig. 5) in the melanotrope subpopulations was studied by confocal microscopy. An initial visual inspection revealed that for all antisera tested, immunofluorescence exhibited a punctate appearance, which reflects the localization of these granins within secretory granules. A more detailed analysis showed that the distribution of the immunoreactive signal slightly differed for the distinct antibodies employed. In particular, immunolabeling for the SgII-derived peptide SN was concentrated in the periphery of cells, whereas immunoreaction against the CgA-related peptide EL35 was stronger in perinuclear regions. In contrast, immunofluorescence to WE14 and EM66, which label CgA and SgII, respectively, was homogeneously distributed throughout the cytoplasm.
There were no apparent differences between the distribution of the immunocytochemical staining in storage melanotropes and that in secretory melanotropes. In contrast, differences in staining intensity between melanotrope subtypes were evident. Accordingly, quantification of immunofluorescence signals in single cells showed that immunoreactivity to CgA, as revealed by either anti-EL35 or anti-WE14 antiserum, was higher in storage cells than in secretory melanotropes (Fig. 6, A and B). Conversely, secretory cells showed higher immunoreactive levels of the two SgII-derived peptides, SN and EM66, than storage melanotropes (Fig. 6, C and D). Taken together, data from immunocytochemistry support the view that although CgA is more abundant in the inactive, storage melanotropes, SgII predominates in the highly active secretory melanotropes.
Discussion
In the present study we aimed at gaining additional insight into the cellular and physiological roles of two major members of the granin family, CgA and SgII, by establishing the basic features of their expression, subcellular distribution, and processing in pituitary melanotropes in relation to the secretory activity of the cells.
Semiquantitative RT-PCR of neurointermediate lobe extracts showed that frog melanotropes express CgA mRNA, thus confirming and extending previous data obtained by in situ hybridization (43). Furthermore, Western blot analysis together with confocal studies demonstrate, for the first time, that CgA is present in amphibian melanotropes. Likewise, we have shown that CgA is proteolytically processed in this cell type, which suggests that this protein may function as a prohormone in the intermediate lobe of amphibians. In line with this observation, Montero-Hadjadje and co-workers (20) reported the presence of the CgA-derived peptides, EL35 and WE14, in rat intermediate lobe extracts by means of HPLC analysis combined with RIA detection using the same antisera employed herein. Nevertheless, although other forms immunoreactive to either anti-EL35 or anti-WE14 were also resolved in the rat, no information was provided about the molecular characteristics of such forms. In this study we have demonstrated that at least four different CgA-immunoreactive fragments, with molecular masses ranging between 94 and 30 kDa, are generated in frog melanotropes, which largely agrees with immunoblot data obtained from whole rat pituitary extracts (20), bovine anterior pituitary vesicles, and mouse AtT-20 cells (44). Taken together, these results indicate that in melanotropes, as in other pituitary cell types, CgA is extensively processed. As a matter of fact, the 94-kDa band, which has also been observed in AtT20 cells by pulse-chase analysis (44) and probably corresponds to the unprocessed protein, was rarely detected in frog melanotrope cell extracts. Furthermore, when present, it exhibited a weak immunostaining signal compared with that shown by the other CgA-derived fragments, suggesting that CgA is rapidly cleaved to lower molecular mass forms.
Based on the different immunoreactive fragments detected with the two anti-CgA antisera employed in this study and the potential dibasic cleavage sites present in the frog CgA sequence (43), and taking into account that CgA processing can proceed from both the C- and the N-terminal ends of the protein (44, 45, 46), we propose a theoretical working model of processing of CgA in melanotropes (Fig. 7). Specifically, CgA proteolysis would start at the N terminus of the protein, at vasostatin I or II cleavage sites (Lys77-Lys78 and Lys119-Arg120, respectively), generating the 67-kDa fragment revealed with antisera against the peptides EL35 and WE14, which are both located at the C-terminal moiety of the protein. Additional cleavage of CgA would occur at the C terminus, most likely at Arg342-Lys343 to give rise to the C-terminal peptide EL35, because the resulting 45-kDa fragment is exclusively detected by the anti-WE14 serum. Next, cleavage at either the C-flanking region of WE14 (at Lys291-Arg292) or, alternatively, at the N terminus of the 45-kDa fragment would generate the smallest, 30-kDa form.
Similar to that found for CgA, we observed that frog melanotropes also express SgII mRNA and process the resulting protein to smaller peptides. Specifically, the two antibodies used in the present study, anti-SN and anti-EM66, revealed the existence of seven different immunoreactive fragments with molecular masses ranging between 97 and 30 kDa and with somewhat variable abundance. The band with the highest molecular mass is comparable to that detected by SDS-PAGE in extracts from human adrenal gland (23) and fetal pituitary (47) and probably corresponds to the uncleaved precursor despite the fact that its apparent molecular mass is higher than that reported for this protein in Xenopus intermediate lobe (25). In support of this, the observed molecular mass of frog SgII by SDS-PAGE is, as occurred for CgA, higher than its theoretical molecular mass (67 and 44 kDa for frog SgII and CgA, respectively) (28, 43), a phenomenon that has been proposed to be related to the acidic nature of granins as well as to the occurrence of posttranslational modifications (i.e. glycosylation, phosphorylation, and sulfation) of these proteins (23, 48, 49).
According to our present results as well as to those reported for Xenopus intermediate lobe (25) and other mammalian neuroendocrine cells (22, 50, 51, 52), which indicate that SgII proteolytic cleavage proceeds from the C terminus to the N-terminal end of the protein, we propose a possible pattern of sequential processing of SgII in frog melanotropes, which is depicted in Fig. 8. It is worth mentioning that melanotropes from both Rana and Xenopus contain some intermediate SgII-processing products (i.e. 55-kDa fragment in frog) that are not present in neuroendocrine cell types of other species even when using the same antisera as those employed in this study (23). Although this may be due to cell-specific differences in SgII processing (30, 31), it may also be related to the existence of additional dibasic cleavage sites in the sequence of amphibian SgII (28, 53).
The antibodies used for the characterization of frog SgII were expected to provide a similar pattern of immunoreactive bands, because their corresponding antigens, the peptides SN and EM66, are located adjacent in the sequence of the protein. However, only 66- and 32-kDa peptides were recognized by both antisera. Moreover, our immunocytochemical studies show that the immunofluorescence signal to anti-SN was preferentially located in the periphery of melanotropes, whereas immunostaining for the anti-EM66 serum was more homogeneously distributed within the cells. Taken together, these results suggest the existence of temporal and/or spatial changes in the structural organization of SgII, probably related to its processing and trafficking through the regulated secretory pathway, which result in differences in the affinities of anti-EM66 and anti-SN for the different fragments sequentially generated from this protein.
Interestingly, our data also revealed that regardless of its relative abundance, the pattern of proteolytic cleavage of either CgA or SgII seems to be specific for the melanotrope cell subtype. In particular, storage melanotropes preferentially contain larger CgA- and SgII-derived fragments, whereas the shorter immunoreactive forms predominate in secretory melanotropes, thus demonstrating that the extent of granin processing is correlated to the secretory activity of the cells. Because it has been shown that the proprotein convertases PC1 and PC2 participate in the proteolytic processing of CgA and SgII (44, 50, 54, 55), as it is also the case for POMC to generate -MSH (reviewed in Ref.56), it is tempting to propose that these enzymes may be more active and/or abundant in secretory melanotropes. Studies are underway in our laboratory aimed at exploring this possibility.
To characterize CgA and SgII in relation to the secretory status of cells, we analyzed the expression and protein content of both granins in the two subtypes of melanotropes. Given the proposed role of granins in promoting granulogenesis (10, 21, 57, 58), we expected both CgA and SgII to be more abundantly expressed in the melanotropes exhibiting higher POMC mRNA content and -MSH secretory rate, that is, in the active secretory melanotropes. In fact, although secretory melanotropes show a low amount of secretory granules in electron microscopic images (34), they exhibit a 3-fold higher -MSH secretory rate than the highly granulated, storage melanotropes (34, 35, 36, 37, 38), thus indicating a rapid formation and/or transit of granules in this active secretory melanotrope cell subset. Accordingly, SgII mRNA levels were indeed significantly higher in secretory melanotropes than in storage cells. We have also shown that secretory melanotropes from frog exhibit a stronger immunofluorescence signal to SN or EM66 than storage melanotrope counterparts, which is suggestive of higher SgII protein content in this cell subset. These results suggest that SgII may be important to sustain a highly secretory state in melanotropes, which is in agreement with the proposed role for SgII in secretory granule biogenesis (21, 58). Consistent with this idea is the observation that SgII is expressed in coordination with POMC in Xenopus intermediate lobe under different experimental conditions in vivo in relation to the amount of -MSH produced by the gland (53). Alternatively, SgII may act as an assembly factor to facilitate sorting and storage of POMC-derived products in secretory granules, as it has been previously proposed for pituitary LH (59). These two, nonmutually exclusive functions have been also suggested for CgA (10, 60, 61). For this reason, it was surprising that both subtypes of melanotropes exhibited similar CgA mRNA levels. Furthermore, immunochemical data showed that at the protein level, storage melanotropes contained more CgA than secretory cells, a difference that is probably attributable to the higher number of CgA-immunolabeled secretory granules found in the former. In view of the low biosynthetic and secretory activities of storage melanotropes both under basal conditions and in response to stimulatory factors (TRH) (34, 35), it seems reasonable to propose that, as occurs for the melanotropic hormone, the high CgA content found in these cells is due to their ability to store higher amounts of secretory granules, rather than to a possible high biosynthetic rate of this granin. In light of our findings that, contrary to SgII, CgA expression or protein content does not show a clear, direct correlation with the degree of secretory activity of melanotropes, it is tempting to speculate that this granin, at least in this cell type, does not play a primary role as a promoter of -MSH release, but may work as a permissive element in the formation and/or storage of secretory granules. In line with these findings, studies using the LT2 gonadotrope cell line demonstrated that CgA production and LH release are inversely correlated, whereas LH storage and release are closely associated with parallel changes in SgII (62). Hence, when viewed together, these and our present results suggest that CgA and SgII play distinct, albeit probably complimentary, roles in the secretory pathway of endocrine cells.
Footnotes
This work was supported by grants from Institut National de la Sante et de la Recherche Medicale, Unite 413, Junta de Andalucia (CVI-0139), the Conseil Regional de Haute-Normandie, and the Ministerio de Educacion y Ciencia (BFI-2001-2007, BFU2004-03883). J.R.P. was the recipient of a European Science Foundation grant. H.V. was the recipient of the 2003 Betancourt-Perronet Price from the Ministerio de Educacion y Ciencia (Spain).
First Published Online December 15, 2005
Abbreviations: CgA, Chromogranin A; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; POMC, proopiomelanocortin; SgII, secretogranin II; SN, secretoneurin.
Accepted for publication November 29, 2005.
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Immunology, University of Cordoba (J.R.P., R.V.-M., D.C.G., A.R.-N., F.G.-N., J.P.C., M.M.M.), 14014 Cordoba, Spain
Institut National de la Sante et de la Recherche Medicale, Unite 413, European Institute for Peptide Research, Laboratory of Cellular and Molecular Neuroendocrinology, Unite Associee Centre National de la Recherche Scientifique, University of Rouen (Y.A., M.C.T., H.V.), 76821 Mont Saint Aignan, France
Abstract
Chromogranin A (CgA) and secretogranin II (SgII) are neuroendocrine secretory proteins that participate in regulation of the secretory pathway and also serve as precursors of biologically active peptides. To investigate whether there is a relationship between the expression, distribution, and processing of CgA and SgII and the degree of secretory activity, we employed two melanotrope subpopulations of the pituitary intermediate lobe that exhibit opposite secretory phenotypes. Thus, although one of the melanotrope subtypes shows high secretory activity, the other exhibits characteristics of a hormone storage phenotype. Our data show that SgII expression levels were higher in secretory melanotropes, whereas CgA expression showed similar rates in both cell subsets. The use of various antibodies revealed the presence of the unprocessed proteins as well as three CgA-derived peptides (67, 45, and 30 kDa) and six SgII-derived peptides (81, 66, 55, 37, 32, and 30 kDa) in both subpopulations. However, the smallest molecular forms of both granins predominated in secretory melanotropes, whereas the largest SgII- and CgA-immunoreactive peptides were more abundant in storage melanotropes, which is suggestive of a more extensive processing of granins in the secretory subset. Confocal microscopy studies showed that CgA immunoreactivity was higher in storage cells, but SgII immunoreactivity was higher in secretory melanotropes. Taken together, our results indicate that SgII and CgA are differentially regulated in melanotrope subpopulations. Thus, SgII expression is strongly related to the secretory activity of melanotrope cells, whereas CgA expression may not be related to secretory rate, but, rather, to hormone storage in this endocrine cell type.
Introduction
CHROMOGRANIN A (CgA) and secretogranin II (SgII) are members of the chromogranin/secretogranin family of proteins, collectively referred to as granins. The granin family also includes chromogranin B (CgB; also known as SgI), secretogranin III (SgIII/1B 1075), secretogranin IV/HISL-19 antigen (SgIV), and 7B2 (also known as secretogranin V; SgV) (1, 2). More recently, three new proteins, BRCA1 (3), NESP55 (4), and pro-SAAS (5), have also been proposed to belong to this family. Typically, granins are highly acidic proteins with an exclusive expression in endocrine, neuroendocrine, and neuronal cells. They are major constituents of secretory granules, in which they are costored with hormones and other products of the regulated secretory pathway (1, 6). Granins have been proposed to participate in the sorting and packaging of hormones and peptide precursors into secretory granules (7), yet the precise role of each granin within the secretory pathway is not completely understood. In addition to their intracellular functions, granins contain multiple dibasic amino acid cleavage sites and are considered precursors of peptides with autocrine, paracrine, or endocrine actions (8).
CgA was the first member of this family to be identified (9), and despite being the most studied granin, its precise function in neurosecretion remains controversial. Thus, although some researchers propose that CgA acts as an on/off switch, controlling secretory granule formation (10), others suggest that it may participate as a simple cargo protein (11) or act as an assembly factor of newly synthesized proteins (12). As a prohormone, CgA is the precursor of several active peptides, including the 14-amino-acid peptide WE14, which modulates histamine secretion in mast cells (13). Pancreastatin, vasostatin I and II, catestatin, parastatin, and chromacin are other CgA-derived peptides with known biological activities (Refs. 14, 15, 16, 17, 18 ; reviewed in Ref.19). Another potential peptide contained in the CgA sequence is the C-terminal 35-amino-acid fragment EL35, which is present in rat pituitary and adrenal glands (20).
SgII has been proposed to act as an assembly factor or helper of granule formation based on its ability to generate granule-like structures when expressed in cell lines lacking a regulated secretory pathway (21). SgII is processed in different neuroendocrine tissues (22, 23, 24, 25, 26), although only the 33-amino-acid-derived peptide secretoneurin (SN) has been shown to have biological activity (reviewed in Ref.27). Other fragments contained in the SgII sequence that are flanked by dibasic sites are EM66, which corresponds to a highly conserved region of the protein (28), and the recently identified peptide manserin (29).
As is the case for other protein precursors, the extent of cleavage of granins and, thus, the number and type of derived peptides are tissue and species specific (20, 30, 31). Additionally, granin expression is regulated in a cell-specific manner in response to a variety of stimuli (reviewed in Ref.32). In this study we attempted to characterize the expression, processing pattern, and intracellular distribution of CgA and SgII in relation to secretory activity in normal, nontumoral endocrine cells. To this end, we used melanotropes of the frog pituitary intermediate lobe as a cellular model. Melanotropes synthesize the precursor protein proopiomelanocortin (POMC), which is proteolytically processed in these cells to -MSH (33). Detailed ultrastructural and functional analyses of melanotropes have revealed that these cells can acquire two opposite secretory phenotypes, high secretory and biosynthetic activity (i.e. active secretory phenotype) or, alternatively, hormone storage (i.e. hormone storage phenotype), that coexist in the gland as two distinct cell subtypes containing a low amount of secretory granules (secretory melanotropes) or a highly granulated cytoplasm (storage melanotropes), respectively (34, 35, 36, 37, 38, 39, 40). To be more specific, although secretory melanotropes show low intracellular -MSH content, but display high POMC mRNA levels and -MSH secretory rate, storage melanotropes exhibit high -MSH intracellular levels, but low POMC mRNA content and -MSH release (34, 35, 36, 37, 38, 39, 40). Armed with this powerful model, we explored the possible relationship between these opposite secretory phenotypes and several features of CgA and SgII. Interestingly, we observed that although both CgA and SgII are expressed and processed in both melanotrope subtypes, the degree of granin processing was strongly dependent on the secretory status of the cells. Moreover, the two granins are differentially regulated in secretory and storage melanotropes at both the mRNA and protein level, suggesting distinct intracellular roles for CgA and SgII in the functioning of the regulated secretory pathway.
Materials and Methods
Reagents
The following materials were obtained from the indicated sources: Percoll and the ECL Plus kit were purchased from Amersham Biosciences (Barcelona, Spain); RNA-easy Mini Kit columns and ribonuclease-free deoxyribonuclease from Qiagen (Hilden, Germany); SuperScript II, Taq-DNA polymerase, ribonuclease inhibitor, deoxy-NTP mixture, and goat antirabbit conjugated to Alexa 594 from Molecular Probes (Barcelona, Spain); the protease inhibitors chymostatin, leupeptin, antipain, and pepstatin A and 5-bromo-4-chloro-3-indolyl phosphate from Roche (Barcelona, Spain). All other reagents were purchased from Sigma-Aldrich Corp. (Madrid, Spain).
Animals
All animal manipulations were performed according to the recommendations of the local ethical committees and under the supervision of authorized investigators. Adult frogs (Rana ridibunda) of about 40 g body weight (Ranas Orense, Mosqueiro, Spain) were maintained at 8 C on a 12-h light, 12-h dark photoperiod for at least 1 wk before death. The animals were killed by decapitation, and neurointermediate lobes were carefully dissected under a microscope. Glands were transferred to sterile Leibovitz culture medium (L-15) diluted 2:3 to adjust to R. ridibunda osmolality and supplemented with 1 mM glucose and 0.4 mM CaCl2 (pH 7.4). For mRNA and protein extractions, samples were frozen in liquid nitrogen and stored at –80 C until use.
Cell dispersion and isolation of melanotrope cell subtypes
Neurointermediate lobes (n = 30–40/experiment) were enzymatically and mechanically dispersed as described previously (34) until a homogeneous cellular suspension was obtained. For separation of the two melanotrope subsets, dispersed cells (1–2 x 106 cells in 250 μl culture medium) were layered on a 9-ml hyperbolic density gradient (1.027–1.072 g/ml) of Percoll (34). After centrifugation of the gradient (3000 x g, 25 min, 4 C), nine fractions of 1 ml each were collected manually. Fractions 5–7 contained secretory melanotropes (low-density cells), and fraction 1 contained storage melanotropes (high-density cells). The proportions of melanotropes recovered from these fractions were comparable to those previously reported (34, 35).
RNA isolation
Total RNA from whole cell populations of frog neurointermediate lobe or from the separate melanotrope subsets was isolated using RNA-easy Mini Kit columns followed by ribonuclease-free deoxyribonuclease treatment. The concentration and purity of the RNA samples were determined by UV spectroscopy at 260/280 nm.
Semiquantitative RT-PCR
The expression levels of CgA and SgII mRNAs in the two melanotrope cell subtypes were measured by semiquantitative RT-PCR, using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a housekeeping gene. Primers for PCR amplification were designed with Oligo software (National Biosciences, Cascade, CO) based on the corresponding cDNA sequences from R. ridibunda as follows: GAPDH sense, 5'-TTTCACCGCTACACAGAAG-3'; and GAPDH antisense, 5'-GTTGCTGTAACCGAATTCA-3' for the amplification of a 426-bp fragment of GAPDH cDNA; CgA sense, 5'-CGAGGAGATGAAAGGATTA-3'; and CgA antisense, 5'-ATCCTCATCGAATTGTCCT-3' for the amplification of a 574-bp fragment of CgA cDNA; and SgII sense, 5'-AGGCCAAGCAAACAACAAG-3'; and SgII antisense, 5'-CACCAAACTTCCTGCCATC-3' for the amplification of a 502-bp fragment of SgII cDNA.
RT and PCR were run in two separate steps. To enable appropriate amplification in the exponential phase for each target gene, PCR amplifications of GAPDH, CgA, and SgII transcripts were carried out in separate reactions with a different number of cycles (see Results), using the corresponding cDNA templates generated in single RT reactions. Briefly, equal amounts of total RNA (0.5 μg) from frog neurointermediate lobes or from the separate melanotrope subsets were heat denatured and reverse transcribed by incubation at 42 C for 60 min with 200 U SuperScript II, 40 U ribonuclease inhibitor, 10 μM deoxy-NTP mixture, and 250 ng random hexamer primers in a final volume of 40 μl 1x SuperScript II buffer. The reactions were terminated by heating at 75 C for 5 min and cooling on ice. For the optimization of the semiquantitative RT-PCR procedure, different concentrations of cDNA obtained from dispersed neurointermediate lobes and different number of cycles were tested for each gene to ensure amplification in the exponential phase. The reactions were carried out in a final volume of 50 μl 1x PCR buffer in the presence of 5 U Taq-DNA polymerase, 10 μM deoxy-NTP mixture, and the appropriate primer pairs (10 μM of each primer). PCRs consisted of, firstly, a denaturing cycle at 95 C for 5 min, followed by the number of cycles selected for optimal amplification of each gene, 30 sec at 95 C for denaturation, annealing at 55 C, and an extension cycle at 72 C for 1 min. A final extension cycle at 72 C for 7 min was included. PCR products were quantified at the end of amplification by electrophoresis on agarose gel (1%) containing ethidium bromide and measurement of signal intensity with Quantity-One software (Bio-Rad Laboratories, Inc., Hercules, CA). mRNA levels of CgA and SgII in each sample were calculated as a ratio of that for GAPDH in the same sample. The specificity of the PCR procedure was checked by omission of the cDNA template in the amplification reaction.
Western blot analysis
The study by Western blotting of CgA and SgII proteins in melanotrope cell extracts was carried out according to the method of Laemmli (41) with the appropriate modifications. In brief, cells were resuspended in 0.01 M PBS (pH 7.4) containing 20 μg/ml of the protease inhibitors chymostatin, leupeptin, antipain, and pepstatin A; sonicated (30 sec); and centrifuged at 3000 rpm for 5 min at 4 C to remove undisrupted cells and debris. Before electrophoresis, cellular extracts (20 μg protein) were suspended in 5x Laemmli buffer [62.5 mM Tris, 12.5% glycerol, 1.25% sodium dodecyl sulfate, 2.5% -mercaptoethanol, and 0.0125% bromophenol blue (pH 7.5)]. After boiling for 5 min, samples were separated by SDS-PAGE (10% acrylamide) using the Protean II Cell System (Bio-Rad Laboratories, Inc.) and blotted onto nitrocellulose membranes. Blots were stained with Ponceau S for visualization of protein bands and to confirm equal protein loading for further comparative analysis. Then blots were destained, blocked during 2 h in blocking buffer [5% low-fat milk in Tris-buffered saline (pH 7.6) with 0.05% Tween 20], and incubated overnight at 4 C with the corresponding specific antibodies. Two distinct regional-specific polyclonal rabbit antisera to human CgA (20) and SgII were used (23). Specifically, the antibodies were directed against the 14-amino-acid CgA-derived peptide WE14 (CgA273–285) and the C-terminal 35-amino-acid peptide EL35 (CgA344–378) for the analysis of CgA, and against SN and SgII187–252 (EM66) for the determination of SgII. Blots were then incubated for 1 h with a goat antirabbit IgG secondary antibody coupled to alkaline phosphatase. Visualization of the antigen/antibody complex was carried out by immersion of the blots in developing buffer [100 mM Tris, 100 mM NaCl, and 5 mM MgCl2 (pH 9.0)] containing nitro blue tetrazolium salt (7.5%) and 5-bromo-4-chloro-3-indolyl phosphate (5%). After immunostaining for granins, blots were allowed to dry, and the intensities of the immunoreactive bands were measured (see below). Thereafter, blots were subjected to a second immunostaining sequence using anti--actin and a horseradish peroxidase-conjugated secondary antibody. Blots were then revealed by chemiluminescence with the ECL Plus kit according to the manufacturer’s instructions. Signal intensities of immunoreactive bands were evaluated with the Quantity-One software described above. Quantitative data from the immunoreactive bands revealed with the anti-CgA anti-SgII sera were normalized against the corresponding -actin values measured on the same blots.
As a control of the specificity of the immunoreaction, blots from samples run in parallel were incubated in blocking reagent alone under the same conditions as those incubated with each specific antiserum. All cell extracts gave negative results when the primary antibodies were omitted.
Confocal microscopy
After dispersion of frog neurointermediate lobes and separation of the melanotrope subpopulations in density gradients, cells were washed in PBS. Fifty-microliter aliquots containing 40,000 cells were plated on slides. Cells were fixed in 4% paraformaldehyde for 15 min, rinsed in PBS, and incubated in the same buffer containing 1% BSA and 0.3% Triton X-100 for 1 h. Thereafter, cells were incubated overnight at 4 C in a humid atmosphere with anti-EL35 or anti-WE14 (CgA) and anti-EM66 or anti-SN (SgII) antibodies diluted in PBS containing 0.3% Triton X-100 and 0.5% BSA at a final dilution of 1:1000. The slides were then rinsed in PBS and incubated for 2 h with secondary antibody conjugated to Alexa 594 at a 1:500 dilution. Finally, the sections were rinsed in PBS and mounted with PBS/glycerol (vol/vol). The preparations were examined under a TCS-SP2-AOBS confocal laser scanning microscope attached to a Leica DM IRE2 inverted epifluorescence microscope equipped with a DMRXA2 lamp (Leica Corp., Heidelberg, Germany). To study the specificity of the immunoreaction, the following controls were performed: 1) omission of the specific antiserum, and 2) incubation with nonimmune rabbit serum instead of the primary antibodies.
The intensity of fluorescence signals in single secretory or storage melanotropes was monitored using an epifluorescence microscope (Eclipse TE2000-E, Nikon, Tokyo, Japan) fitted with a Plan-Fluor x60 oil immersion objective. The slides were epiilluminated at 540 nm for 800 msec for the acquisition of images of single cells. Image acquisition was controlled using Metafluor PC software (Universal Imaging Corp., West Chester, PA), and fluorescence emissions were captured using a CCD camera (C-4880-80, Hamamatsu, Hamamatsu City, Japan) running in 1-bit mode. The images acquired were transferred to off-line storage for subsequent analysis. Regions of the same field devoid of cells were selected for monitoring of the background.
Statistical analysis
For mRNA determinations, three independent RT-PCR experiments using RNA from different extractions were carried out for each target gene. For Western blot studies, three or four independent experiments using protein samples from different extractions were employed for the analysis of each granin. Results are expressed as normalized ratios by using GAPDH (for PCR studies) or -actin (for Western blot analysis) as reference. Data are expressed in arbitrary units (mean ± SEM). All comparisons were made between samples electrophoresed on the same gel and, in the case of the proteins, also blotted onto the same membrane. For analysis of the immunocytochemical signal in single cells to the four different antibodies, at least 20 storage melanotropes or secretory melanotropes obtained from four separate experiments were used. Statistical analysis was carried out with a paired t test (for RT-PCR and Western blot data) or Mann-Whitney rank-sum test (for immunofluorescence measurement in single cells) using the software package Statistica (StatSoft, Inc., Tulsa, OK). Differences were considered significant at P < 0.05.
Results
Semiquantitative RT-PCR analysis of CgA and SgII mRNA expression
The expression levels of CgA and SgII in melanotropes were evaluated by semiquantitative RT-PCR, using GAPDH as an internal standard. The technique was optimized for both transcripts by determining the number of PCR cycles and the amount of cDNA necessary for a quantitative amplification (i.e. within the exponential phase of PCR). Specifically, results from three separate experiments showed that 27 and 30 cycles of PCR amplification, corresponding to the linear portion of the curves for CgA and SgII, respectively, and 1 μl cDNA (equivalent to 25 ng total RNA), provided accurate amplification of both transcripts in mRNA samples from frog neurointermediate lobe and were therefore chosen for subsequent PCR quantifications. It should also be noted that the expression level of GAPDH provides an appropriate endogenous control, because its mRNA levels did not vary between the melanotrope cell subtypes (Fig. 1). No amplification products were obtained when the cDNA template was omitted from the PCR (data not shown).
Transcripts for both CgA (Fig. 1A) and SgII (Fig. 1C) were detected in mRNA extracts from the separate melanotrope cell subpopulations. Quantification of the 574-bp band corresponding to CgA revealed no differences in CgA mRNA content between the two melanotrope subtypes (Fig. 1B). In contrast, secretory melanotropes contained higher SgII mRNA levels than storage melanotropes (Fig. 1D).
Characterization of CgA in melanotropes
The analysis of CgA in frog melanotropes was carried out using two different antibodies: anti-EL35 and anti-WE14, which recognize the C-terminal end of CgA and a fragment of 14 amino acids located in a more internal region of the protein, respectively (20). Results were expressed as normalized ratios using -actin as the reference protein (Fig. 2).
Use of the EL35 antiserum revealed a single band of 67 kDa, which was apparent in extracts from both storage and secretory melanotropes (Fig. 2A). However, densitometric quantification of the band obtained for each melanotrope cell subtype showed a significantly stronger immunoreaction in extracts from storage melanotropes than from secretory cells (Fig. 2B). Likewise, the antibody against WE14 also labeled a 67-kDa band (Fig. 2C), which was more abundant in the storage cell subset than in secretory melanotropes (Fig. 2D). In addition, the WE14 antiserum revealed the presence of two additional fragments (45 and 30 kDa), which were differentially distributed in the two melanotrope cell subsets (Fig. 2C). Thus, although the smallest molecular form was hardly detectable in storage melanotropes, the intermediate mass band of 45 kDa, which displayed the strongest immunoreaction among the three bands appearing in the melanotrope subsets, clearly predominated in storage cells over secretory melanotropes (Fig. 2D). Finally, a weak immunostained band of 94 kDa was sometimes detected in extracts from storage cells (Fig. 2C).
Characterization of SgII in melanotropes
The antibodies used for this study, anti-SN and anti-EM66, recognize adjacent regions in the SgII sequence separated by a dibasic cleavage site (23, 42). These antibodies detected both common and distinct immunoreactive bands in melanotropes (Figs. 3 and 4). Specifically, anti-SN revealed a band of high molecular mass (66 kDa) as well as three bands of lower molecular masses (37, 32, and 30 kDa) in extracts from secretory and storage melanotropes (Fig. 3A). However, their relative abundance differed between the two melanotrope cell subtypes (Fig. 3B). Thus, densitometric quantification of the immunoreactive bands obtained for each cell subset and subsequent normalization of these values using -actin as a reference revealed that although the 66-kDa fragment was the most prominent SgII-derived cleavage product in storage melanotropes, the smallest, 30-kDa peptide predominated in the highly active cell subset. Immunostaining of both 37- and 32-kDa bands was numerically higher in secretory melanotropes than in storage melanotropes, although these differences did not reach statistical significance.
In addition to the 66- and 32-kDa bands, the antibody against the EM66 sequence also detected the presence of three other fragments of 97, 81, and 55 kDa in extracts from the two melanotrope cell subtypes (Fig. 4A). Occasionally, a weak immunopositive band of 30 kDa was observed in secretory melanotrope extracts (data not shown). Densitometric quantification showed no differences between storage and secretory melanotropes in the relative amount of either the two higher molecular mass fragments or in the 55-kDa fragment (Fig. 4B). However, in accordance with that found for the anti-SN antiserum, the 66-kDa form predominated in the melanotropes displaying low synthetic and secretory activities (Fig. 4B). In contrast, secretory melanotropes contained higher levels of the smallest molecular form of 32 kDa (Fig. 4B).
Confocal microscopy
Cellular distribution of CgA and SgII (Fig. 5) in the melanotrope subpopulations was studied by confocal microscopy. An initial visual inspection revealed that for all antisera tested, immunofluorescence exhibited a punctate appearance, which reflects the localization of these granins within secretory granules. A more detailed analysis showed that the distribution of the immunoreactive signal slightly differed for the distinct antibodies employed. In particular, immunolabeling for the SgII-derived peptide SN was concentrated in the periphery of cells, whereas immunoreaction against the CgA-related peptide EL35 was stronger in perinuclear regions. In contrast, immunofluorescence to WE14 and EM66, which label CgA and SgII, respectively, was homogeneously distributed throughout the cytoplasm.
There were no apparent differences between the distribution of the immunocytochemical staining in storage melanotropes and that in secretory melanotropes. In contrast, differences in staining intensity between melanotrope subtypes were evident. Accordingly, quantification of immunofluorescence signals in single cells showed that immunoreactivity to CgA, as revealed by either anti-EL35 or anti-WE14 antiserum, was higher in storage cells than in secretory melanotropes (Fig. 6, A and B). Conversely, secretory cells showed higher immunoreactive levels of the two SgII-derived peptides, SN and EM66, than storage melanotropes (Fig. 6, C and D). Taken together, data from immunocytochemistry support the view that although CgA is more abundant in the inactive, storage melanotropes, SgII predominates in the highly active secretory melanotropes.
Discussion
In the present study we aimed at gaining additional insight into the cellular and physiological roles of two major members of the granin family, CgA and SgII, by establishing the basic features of their expression, subcellular distribution, and processing in pituitary melanotropes in relation to the secretory activity of the cells.
Semiquantitative RT-PCR of neurointermediate lobe extracts showed that frog melanotropes express CgA mRNA, thus confirming and extending previous data obtained by in situ hybridization (43). Furthermore, Western blot analysis together with confocal studies demonstrate, for the first time, that CgA is present in amphibian melanotropes. Likewise, we have shown that CgA is proteolytically processed in this cell type, which suggests that this protein may function as a prohormone in the intermediate lobe of amphibians. In line with this observation, Montero-Hadjadje and co-workers (20) reported the presence of the CgA-derived peptides, EL35 and WE14, in rat intermediate lobe extracts by means of HPLC analysis combined with RIA detection using the same antisera employed herein. Nevertheless, although other forms immunoreactive to either anti-EL35 or anti-WE14 were also resolved in the rat, no information was provided about the molecular characteristics of such forms. In this study we have demonstrated that at least four different CgA-immunoreactive fragments, with molecular masses ranging between 94 and 30 kDa, are generated in frog melanotropes, which largely agrees with immunoblot data obtained from whole rat pituitary extracts (20), bovine anterior pituitary vesicles, and mouse AtT-20 cells (44). Taken together, these results indicate that in melanotropes, as in other pituitary cell types, CgA is extensively processed. As a matter of fact, the 94-kDa band, which has also been observed in AtT20 cells by pulse-chase analysis (44) and probably corresponds to the unprocessed protein, was rarely detected in frog melanotrope cell extracts. Furthermore, when present, it exhibited a weak immunostaining signal compared with that shown by the other CgA-derived fragments, suggesting that CgA is rapidly cleaved to lower molecular mass forms.
Based on the different immunoreactive fragments detected with the two anti-CgA antisera employed in this study and the potential dibasic cleavage sites present in the frog CgA sequence (43), and taking into account that CgA processing can proceed from both the C- and the N-terminal ends of the protein (44, 45, 46), we propose a theoretical working model of processing of CgA in melanotropes (Fig. 7). Specifically, CgA proteolysis would start at the N terminus of the protein, at vasostatin I or II cleavage sites (Lys77-Lys78 and Lys119-Arg120, respectively), generating the 67-kDa fragment revealed with antisera against the peptides EL35 and WE14, which are both located at the C-terminal moiety of the protein. Additional cleavage of CgA would occur at the C terminus, most likely at Arg342-Lys343 to give rise to the C-terminal peptide EL35, because the resulting 45-kDa fragment is exclusively detected by the anti-WE14 serum. Next, cleavage at either the C-flanking region of WE14 (at Lys291-Arg292) or, alternatively, at the N terminus of the 45-kDa fragment would generate the smallest, 30-kDa form.
Similar to that found for CgA, we observed that frog melanotropes also express SgII mRNA and process the resulting protein to smaller peptides. Specifically, the two antibodies used in the present study, anti-SN and anti-EM66, revealed the existence of seven different immunoreactive fragments with molecular masses ranging between 97 and 30 kDa and with somewhat variable abundance. The band with the highest molecular mass is comparable to that detected by SDS-PAGE in extracts from human adrenal gland (23) and fetal pituitary (47) and probably corresponds to the uncleaved precursor despite the fact that its apparent molecular mass is higher than that reported for this protein in Xenopus intermediate lobe (25). In support of this, the observed molecular mass of frog SgII by SDS-PAGE is, as occurred for CgA, higher than its theoretical molecular mass (67 and 44 kDa for frog SgII and CgA, respectively) (28, 43), a phenomenon that has been proposed to be related to the acidic nature of granins as well as to the occurrence of posttranslational modifications (i.e. glycosylation, phosphorylation, and sulfation) of these proteins (23, 48, 49).
According to our present results as well as to those reported for Xenopus intermediate lobe (25) and other mammalian neuroendocrine cells (22, 50, 51, 52), which indicate that SgII proteolytic cleavage proceeds from the C terminus to the N-terminal end of the protein, we propose a possible pattern of sequential processing of SgII in frog melanotropes, which is depicted in Fig. 8. It is worth mentioning that melanotropes from both Rana and Xenopus contain some intermediate SgII-processing products (i.e. 55-kDa fragment in frog) that are not present in neuroendocrine cell types of other species even when using the same antisera as those employed in this study (23). Although this may be due to cell-specific differences in SgII processing (30, 31), it may also be related to the existence of additional dibasic cleavage sites in the sequence of amphibian SgII (28, 53).
The antibodies used for the characterization of frog SgII were expected to provide a similar pattern of immunoreactive bands, because their corresponding antigens, the peptides SN and EM66, are located adjacent in the sequence of the protein. However, only 66- and 32-kDa peptides were recognized by both antisera. Moreover, our immunocytochemical studies show that the immunofluorescence signal to anti-SN was preferentially located in the periphery of melanotropes, whereas immunostaining for the anti-EM66 serum was more homogeneously distributed within the cells. Taken together, these results suggest the existence of temporal and/or spatial changes in the structural organization of SgII, probably related to its processing and trafficking through the regulated secretory pathway, which result in differences in the affinities of anti-EM66 and anti-SN for the different fragments sequentially generated from this protein.
Interestingly, our data also revealed that regardless of its relative abundance, the pattern of proteolytic cleavage of either CgA or SgII seems to be specific for the melanotrope cell subtype. In particular, storage melanotropes preferentially contain larger CgA- and SgII-derived fragments, whereas the shorter immunoreactive forms predominate in secretory melanotropes, thus demonstrating that the extent of granin processing is correlated to the secretory activity of the cells. Because it has been shown that the proprotein convertases PC1 and PC2 participate in the proteolytic processing of CgA and SgII (44, 50, 54, 55), as it is also the case for POMC to generate -MSH (reviewed in Ref.56), it is tempting to propose that these enzymes may be more active and/or abundant in secretory melanotropes. Studies are underway in our laboratory aimed at exploring this possibility.
To characterize CgA and SgII in relation to the secretory status of cells, we analyzed the expression and protein content of both granins in the two subtypes of melanotropes. Given the proposed role of granins in promoting granulogenesis (10, 21, 57, 58), we expected both CgA and SgII to be more abundantly expressed in the melanotropes exhibiting higher POMC mRNA content and -MSH secretory rate, that is, in the active secretory melanotropes. In fact, although secretory melanotropes show a low amount of secretory granules in electron microscopic images (34), they exhibit a 3-fold higher -MSH secretory rate than the highly granulated, storage melanotropes (34, 35, 36, 37, 38), thus indicating a rapid formation and/or transit of granules in this active secretory melanotrope cell subset. Accordingly, SgII mRNA levels were indeed significantly higher in secretory melanotropes than in storage cells. We have also shown that secretory melanotropes from frog exhibit a stronger immunofluorescence signal to SN or EM66 than storage melanotrope counterparts, which is suggestive of higher SgII protein content in this cell subset. These results suggest that SgII may be important to sustain a highly secretory state in melanotropes, which is in agreement with the proposed role for SgII in secretory granule biogenesis (21, 58). Consistent with this idea is the observation that SgII is expressed in coordination with POMC in Xenopus intermediate lobe under different experimental conditions in vivo in relation to the amount of -MSH produced by the gland (53). Alternatively, SgII may act as an assembly factor to facilitate sorting and storage of POMC-derived products in secretory granules, as it has been previously proposed for pituitary LH (59). These two, nonmutually exclusive functions have been also suggested for CgA (10, 60, 61). For this reason, it was surprising that both subtypes of melanotropes exhibited similar CgA mRNA levels. Furthermore, immunochemical data showed that at the protein level, storage melanotropes contained more CgA than secretory cells, a difference that is probably attributable to the higher number of CgA-immunolabeled secretory granules found in the former. In view of the low biosynthetic and secretory activities of storage melanotropes both under basal conditions and in response to stimulatory factors (TRH) (34, 35), it seems reasonable to propose that, as occurs for the melanotropic hormone, the high CgA content found in these cells is due to their ability to store higher amounts of secretory granules, rather than to a possible high biosynthetic rate of this granin. In light of our findings that, contrary to SgII, CgA expression or protein content does not show a clear, direct correlation with the degree of secretory activity of melanotropes, it is tempting to speculate that this granin, at least in this cell type, does not play a primary role as a promoter of -MSH release, but may work as a permissive element in the formation and/or storage of secretory granules. In line with these findings, studies using the LT2 gonadotrope cell line demonstrated that CgA production and LH release are inversely correlated, whereas LH storage and release are closely associated with parallel changes in SgII (62). Hence, when viewed together, these and our present results suggest that CgA and SgII play distinct, albeit probably complimentary, roles in the secretory pathway of endocrine cells.
Footnotes
This work was supported by grants from Institut National de la Sante et de la Recherche Medicale, Unite 413, Junta de Andalucia (CVI-0139), the Conseil Regional de Haute-Normandie, and the Ministerio de Educacion y Ciencia (BFI-2001-2007, BFU2004-03883). J.R.P. was the recipient of a European Science Foundation grant. H.V. was the recipient of the 2003 Betancourt-Perronet Price from the Ministerio de Educacion y Ciencia (Spain).
First Published Online December 15, 2005
Abbreviations: CgA, Chromogranin A; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; POMC, proopiomelanocortin; SgII, secretogranin II; SN, secretoneurin.
Accepted for publication November 29, 2005.
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