ARTICLE |
Correspondence to: Shigeyasu Tanaka, Dept. of Cell Biology, Inst. for Molecular and Cellular Regulation, Gunma Univ., Showa-machi 3-39-15, Maebashi 371, Japan.
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Summary |
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Using antibodies specific for pro-opiomelanocortin (POMC), amidated joining peptide (JP), and the prohormone convertase PC1, we showed immunocytochemically that PC1 in a corticotrophic tumor cell line, AtT-20, was co-localized either with POMC or with amidated JP in secretory granules, and also confirmed that POMC was cleaved mainly in secretory granules. Analysis using DAMP (3- [2,4-dinitroanilino]-3'-amino-N-methyldipropylamine) as the pH probe suggested a correlation between POMC processing and acidic pH in the secretory granules. Bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase, completely inhibited POMC processing and caused constitutive secretion of the unprocessed precursor. By contrast, chloroquine, a weak base that is known to neutralize acidic organelles, was unable to inhibit POMC processing. Electron microscopic analysis revealed that, in AtT-20 cells treated with bafilomycin A1, the trans-Golgi cisternae were dilated and few secretory granules were present in the cytoplasm. These observations suggest that acidic pH provides a favorable environment for proteolytic processing of POMC by PC1 but is not required, and that integrity of the trans-Golgi network and sorting of POMC into secretory granules are important for POMC processing. (J Histochem Cytochem 45:425-436, 1997)
Key Words: proteolytic processing, pro-opiomelanocortin, secretory granule, acidification, DAMP, bafilomycin A1, immunocytochemistry, prohormone convertase, AtT-20 cell
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Introduction |
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In endocrine and neuronal cells, most peptide hormones and neuropeptides are produced from larger, inactive precursors through limited endoproteolysis at pairs of basic amino acids during their transport along the exocytic pathway. One of the most extensively studied peptide precursors is pro-opiomelanocortin (POMC), from which ACTH, -MSH, ß-endorphin, and their related peptides are produced in anterior pituitary corticotrophs and in melanotrophs in the pars intermedia (for reviews see
-MSH, and ß-endorphin (for reviews see
Previous studies have shown that acidification of the TGN or secretory granules plays an important role in the proteolytic processing of prohormones, including POMC, and in the secretion of prohormone-derived peptides (
To address the above issues, we first examined the subcellular localization of POMC, a processed product (amidated JP), and PC1 in a corticotrophic tumor cell line, AtT-20, using antibodies specific for a POMC cleavage site, amidated JP, and PC1. Second, we examined the relationship between acidification and POMC processing in the secretory granules using a basic congener of dinitrophenol (DNP), DAMP (3-[2,4-dinitroanilino]-3'-amino-N-methyldipropylamine), as the pH probe. Finally, we examined the effects of bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase (V-ATPase) (
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Materials and Methods |
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Cell Culture
AtT-20/D-16 cells were grown in Dulbecco's modified Eagle's medium supplemented with glucose, Hepes (4.76 g/liter), 10% fetal calf serum, penicillin (100K U/liter), and streptomycin (100 mg/liter) at 37C in 5% CO2. For analysis of immunoprecipitation and electron microscopy, cells were seeded at 1 x 106 cells per 3-cm tissue culture dish. For immunofluorescence, cells were seeded at 2 x 103 - 104 cells/ml on poly-L-lysine-coated coverslips. AtT-20 cells were used for all the experiments after a 3-day culture period.
Antibodies
Antibodies used in this study have been prepared in rabbits and characterized as described previously: anti-human ACTH[1-39] serum (
Immunofluorescence
AtT-20 cells were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, and free aldehyde was quenched by incubating the fixed cells with PBS containing 50 mM NH4Cl. To obtain intense immunostaining for PC1 or PC2, the cells were then treated with Gomori's oxidation mixture according to
Immunogold Electron Microscopy
AtT-20 cells were fixed with a mixture of 0.5% glutaraldehyde, 2% paraformaldehyde, and 1% picric acid in 0.1 M cacodylate buffer, pH 7.4, for 2 hr at 4C. The cells embedded in agarose were dehydrated in ethanol, embedded in LR White (London Resin; Basingstoke, UK), and then sectioned on a Reichert Ultracut-E (Reichert-Jung; Vienna, Austria). For immunolabeling of PC1 or PC2, ultrathin sections were subjected to Gomori's oxidation as described above. Double immunogold labeling of the sections with different antibodies was performed as described previously (
Measurement of pH
The pH of the interior organelles in AtT-20 cells was measured by the DAMP method by
pH = 7.0 - log L/N
where N is the density of DAMP-specific gold particles in a pH 7.0 compartment (the nucleus) and L is the density of DAMP-specific gold particles in an organelle. Specimens containing AtT-20 cells from three different culture dishes were used for quantitative analysis. The number of gold particles per organelle was directly calculated from micrographs printed at a final magnification of x 100,000. The area of the organelle was measured with NIH Image software (version 1.47) on an Apple Macintosh computer. Statistical analysis was performed using the Cochran-Cox test. The statistical significance of the correlation between the accumulation of DAMP and either POMC or amidated JP labeling was evaluated using Spearman's rank correlation. p<0.05 was considered significant.
Immunoprecipitation
Immunoprecipitation was performed as described previously (
Conventional Electron Microscopy
AtT-20 cells incubated for either 30 min or 2.5 hr at 37C in the presence or absence of 10 nM bafilomycin A1 or 200 µM chloroquine were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, for 1 hr at 4C and postfixed for 1 hr at 4C in 1% osmium tetroxide, reduced with 1.5% potassium ferrocyanide, in the same buffer. After further washes with the above buffer, the cells were collected and embedded in agarose. The agarose blocks were dehydrated in ethanol, embedded in an Epon/Araldite mixture, and then the ultrathin sections were stained with uranyl acetate and lead citrate.
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Results |
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Conversion of POMC to Mature Peptides Occurs Mainly in the Secretory Granules
To determine the intracellular sites at which POMC processing occurs in AtT-20 cells, we first examined the subcellular distribution of POMC and its processed peptides at the light microscopic level with indirect immunofluorescence. To do this, two antibodies that we had previously prepared were used: anti-POMC (ST-1), which was raised against a synthetic nonapeptide corresponding to the murine ACTH-ß-LPH cleavage site and is able to recognize POMC but not its processed peptides (
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Co-localization of the Prohormone Convertase PC1 with POMC and Amidated JP in the Same Secretory Granules
Although immunocytochemical studies thus far have revealed the localization of prohormone convertases at the cellular level (
Immunofluorescence analysis showed the localization of PC1 in the perinuclear region and at the tips of cellular processes in AtT-20 cells (Figure 2a). Pre-absorption of the anti-PC1 serum with 1 µg/ml of the corresponding antigen peptide eliminated the staining (Figure 2b). No positive immunofluorescence was seen in any cellular compartments when AtT-20 cells were stained with anti-PC2. This was consistent with previous reports showing that PC1, but not PC2, is expressed in this cell line (
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The pH of Secretory Granules is Lower than That of the Golgi Cisternae
To elucidate the relationship between the processing of POMC and the pH of secretory granules in AtT-20 cells, we examined the acidity of granules with the DAMP method. AtT-20 cells were incubated with 60 µM DAMP, fixed with aldehyde fixatives, and embedded in LR White resin. The LR White resin sections were immunolabeled with anti-DNP antibody followed by immunogold reagent. Many gold particles for DAMP were located in the secretory granules, but few were present in the Golgi apparatus, cell nucleus, and mitochondria (Figure 6a and Figure 6b; small gold particles). In sections singly labeled for DAMP, the density of gold particles in the nucleus, the Golgi apparatus, and the mitochondria was 70.0 ± 8.2 (n = 20), 74.5 ± 5.6 (n = 20), and 68.3 ± 10.2 (n = 20) particles/µm2, respectively. No significant difference in the density was observed among these compartments. In contrast, a variable density of DAMP labeling was found in the secretory granules. As shown in Figure 7, the pH values of secretory granules, estimated according to the formula of
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The labeling density of DAMP was lower in POMC-positive secretory granules near the Golgi apparatus than in amidated JP-positive granules at the tips of the cell processes (Figure 6a and Figure 6b). To define more precisely the relationship between the DAMP labeling density and the processing of POMC to amidated JP in secretory granules, we performed quantitative analysis by double labeling for DAMP and either POMC or amidated JP. As shown in Figure 8, the accumulation of DAMP and the degree of POMC processing appeared to be related, with a tendency towards a negative correlation between the labeling density for DAMP and POMC (r = -0.443; p<0.001) and a positive correlation between DAMP and amidated JP (r = 0.463; p<0.001).
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Effect of Bafilomycin A1 on Processing of POMC and the Morphology of Organelles
To investigate further the correlation between the granular pH and the degree of POMC processing, we examined the effect of bafilomycin A1, a V-ATPase inhibitor known to dissipate the pH gradient across organellar membranes. AtT-20 cells were pulse-labeled with [35S]-methionine and chased in the continuous presence or absence of bafilomycin A1, and then the cell lysates and media were immunoprecipitated with anti-human ACTH[1-39] serum. As shown in Figure 9, the proteins present in the pulse-labeled cells were limited to two main bands of 32 and 29 kD, corresponding to the size of glycosylated POMC (
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We then examined the effect of bafilomycin A1 on the morphology of organelles. Untreated AtT-20 cells are typically characterized by a well-developed rough endoplasmic reticulum and a Golgi apparatus consisting of a stack of six or seven cisternae (Figure 10a). A few secretory granules were scattered near the Golgi apparatus, and secretory granules were more numerous at the tips of the cell processes. Figure 10b shows an electron micrograph of AtT-20 cells treated with 10 nM bafilomycin A1 for 2.5 hr. In these cells, many large vacuoles and dilated cisternae were found near the Golgi apparatus. Careful observation revealed that these vacuoles were partially coated with a clathrin-like substance and did not contain any dense core material (Figure 10b and Figure 10c, arrows). Secretory granules were rarely seen in any cytoplasmic region. After as little as 30 min of treatment, the trans-cisternae of the Golgi apparatus were dilated and few secretory granules were observed in the cytoplasm (data not shown).
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When the bafilomycin A1-treated AtT-20 cells were immunolabeled with anti-POMC or anti-amidated JP, labeling for POMC was found in the vacuoles coated with a clathrin-like substance and in the dilated cisternae (Figure 11), but no immunoreactivity for amidated JP was detected in any subcellular compartment (data not shown). These morphological observations substantiate the data from the pulse-chase analysis.
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When the cells treated with bafilomycin A1 were processed for DAMP immunogold labeling, the accumulation of the pH probe in secretory granules was blocked at the background level (the density of gold particles was 93.4 ± 9.2 particles/µm2; n = 50).
Chloroquine Does Not Inhibit Processing of POMC
Previously,
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Discussion |
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With respect to the intracellular compartments in which proteolytic processing of POMC takes place in AtT-20 cells, there have been two immunocytochemical studies. Using an antibody that specifically recognizes a cleavage site of POMC,
Enzymological studies have shown that the prohormone convertases PC1 and PC2 are most active at a relatively acidic pH (pH 5.0-6.5) (
In an attempt to support the above observations, we examined the effect of the V-ATPase inhibitor, bafilomycin A1, on POMC processing. Recently, it was reported that the internal acidic environment in the secretory pathways was generated by a V-ATPase (
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Footnotes |
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1 Present address: Dept. of Regulation Biology, Faculty of Science, Saitama Univ., Urawa 338, Japan.
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Acknowledgments |
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This work was supported by a research grant (No. 0467002) from the Japanese Ministry of Education, Science, and Culture (ST).
Received for publication March 7, 1996; accepted October 30, 1996.
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