ARTICLE |
Correspondence to: Claude Tougard, Groupe de Biologie de la Cellule Neuroendocrine, INSERM U 36, Collège de France, 11 Place Marcelin Berthelot, 75231 Paris Cedex 05, France.
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Summary |
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The prohormone convertases PC1 and PC2 are subtilisin-related endopeptidases that process prohormone and neuropeptide precursors. Using different ultrastructural immunocytochemical approaches, we have investigated their intracellular distribution in a neuroendocrine cell type that has not been examined thus far, the rat anterior pituitary lactotrope. These cells secrete mainly prolactin and also express the neuroendocrine-specific protein secretogranin II, which is considered a peptide precursor. Our study provides evidence for the expression of PC1 and PC2 in rat lactotropes and provides new information on their subcellular localization. Apart from their presence in the secretory granules, PC1 and PC2 displayed different major localization along the secretory pathway. PC1 immunoreactivity was concentrated in the Golgi apparatus, whereas PC2 immunoreactivity was prominent in the rough endoplasmic reticulum (RER). These observations provide morphological support for previous biochemical analysis of proPC1 and proPC2 post-translational processing, which has demonstrated that PC1 exits very rapidly from the RER, whereas PC2 is retained much longer in this compartment. (J Histochem Cytochem 46:101-108, 1998)
Key Words: prohormone convertases, PC1, PC2, rat anterior pituitary, lactotropes, electron microscope, immunoperoxidase, immunogold
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Introduction |
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The last post-translational modifications resulting in the production of active hormones and neuropeptides consist of the endoproteolytic cleavage of the precursors, followed by exoproteolytic cleavage and N-terminal acetylation and/or C-terminal amidation. Two mammalian endoproteases related to Kex2 have recently been cloned and named prohormone convertases 1 and 2 (PC1 and PC2) (
The cloning of these enzymes has provided specific tools that allow the study of their expression in neuroendocrine tissues. In situ hybridization experiments have shown that PC1 is the major PC expressed in the anterior pituitary and that PC2 is also present in substantial amounts in this tissue (
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Materials and Methods |
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Antibodies
We used rabbit polyclonal antibodies directed against the carboxy terminal segment of mPC1 (AbC-mPC1; segment 629-726) (
Pre-embedding Immunoperoxidase Staining
This labeling was performed directly on rat anterior pituitary cells in primary culture as previously described (
Postembedding Immunogold Staining
Small pieces of male Wistar rat anterior pituitaries were fixed with a mixture of 4% paraformaldehyde-0.05% glutaraldehyde in 0.1M cacodylate buffer (pH 7.4) for 2 hr at 4C. After washing, the tissues were treated with 2% uranyl acetate in 50 mM maleate buffer for 2 hr at 4C, partially dehydrated, and embedded in LR White according to
Ultrathin sections were immunostained as previously reported (
Double immunostaining
Double Immunogold Staining.
The double staining was performed on the same face of the grid. After the first complete immunostaining (i.e., incubation with the first specific antibodies and then with 10-nm protein A-gold particles), the grids were incubated for l hr with unconjugated protein A (0.1 mg/ml in TBS) (Sigma; St Louis, MO), as previously described (
Double Pre- and Postembedding Immunostaining. After the pre-embedding immunoperoxidase staining of PC1 or PC2 in rat anterior pituitary cells in primary culture (as described above), ultrathin sections of immunoreactive cells were then immunostained using the postembedding immunogold approach, with antiserum against rat PRL and then with 10-nm protein A-gold particles.
Western Blotting
Rat anterior pituitaries were homogenized at 4C in a lysis buffer (0.01 M PBS, pH 7.4, containing 0.5% sodium deoxycholate and 0.5% Nonidet P40) in the presence of 200 µM PMSF, 1 µM leupeptin, and 1 µM pepstatin. GH3B6 rat tumor PRL cells, cultured for 6 days, were scraped on ice in the same buffer. Lysates were then centrifuged for 10 min at 15,000 x g at 4C and the supernatants were further solubilized with SDS-PAGE sample buffer containing 50 mM DTT. After separation by SDS-PAGE, proteins were electrotransferred to nitrocellulose sheets (Schleicher & Schuell; Keene, NH). After incubation with the specific antibodies, immunoreactive proteins were revealed with alkaline phosphatase-conjugated sheep anti-rabbit IgG antibodies (Promega; Madison, WI).
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Results |
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Using both immunoperoxidase and immunogold labeling, PC1 and PC2 were detected in most of the glandular cells of the rat anterior pituitary in situ and in primary culture. The immunoreactive sites corresponded to the intracellular compartments involved in the secretory pathway. Differences in the intensity of the immunolabeling and in the respective subcellular distribution of the two PCs were conspicuous, even at low magnification, using the pre-embedding approach and immunoperoxidase staining. With this approach and whatever the cell type, the intensity of PC1 labeling was always higher than that of PC2 (compare Figure 1 and Figure 2). PC1-specific immunoperoxidase staining concerned mainly the Golgi stacks, which were conspicuously labeled in all glandular cells. The rough endoplasmic reticulum (RER) cisternae were very slightly labeled (Figure 1). On the contrary, PC2 was detected only in the RER cisternae (Figure 2). The Golgi saccules were not immunostained. With this approach, as has been previously described for secretory products (
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Because the present study was focused on the immunolocalization of the PCs in PRL cells, we performed double immunostaining of either PC1 or PC2 and of PRL to specifically identify the PRL cells. These double immunostainings confirmed that PRL cells expressed both PC1 and PC2. Using a combination of pre- and postembedding approaches and immunoperoxidase to localize the PCs and immunogold to identify PRL cells, intense and preferential distribution of PC1 within the Golgi saccules (Figure 4) and preferential localization of PC2 on the membrane of the flattened RER cisternae (Figure 5), characteristic of PRL cells, were confirmed in PRL cells. The specific hormonal product, visualized with immunogold, was detected on the matrix of immature and mature secretory granules (Figure 4 and Figure 5). The double immunogold staining with anti-PC1 or anti-PC2 and anti-PRL also confirmed that the two PCs are present in at least some PRL secretory granules (Figure 6). Because the gold particles corresponding to immunoreactive PC1 or PC2 were less numerous than those corresponding to immunoreactive PRL, a portion of the PRL secretory granules appeared devoid of detectable PCs.
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The presence of PC1 and PC2, in the rat anterior pituitary and in the GH3B6 rat PRL cell line was confirmed by the immunoblotting analysis. The antibodies specific for the C-terminal segment of either PC1 or PC2 recognized two large bands in both cell extracts, as already described in the literature. The anti-PC1 antibodies detected a 91-kD band that corresponds to the precursor form and an 84-kD band that corresponds to the mature enzyme (Figure 7). The anti-PC2 antibodies also detected two bands of 75 kD and 65 kD that correspond to the precursor form and to the mature enzyme, respectively (Figure 7). However, the intensity of the immunolabeled bands varied in the two cell extracts. Whereas the dominant form of PC2 was the mature enzyme in the anterior pituitary extracts, pro-PC2 and mature PC2 were as intensely labeled in the GH3B6 cell extracts. This difference was not observed for PC1. These discrepancies could be related to the difference in post-translational processing of the two enzymes (see Discussion) and to the difference in the storage compartment of our two cell models. Most of the glandular cells of the anterior pituitary contained many large secretory granules, whereas GH3B6 cells were characterized by very few small secretory granules.
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Discussion |
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In the present work, we provide immunocytochemical evidence for the expression of PC1 and PC2 in rat lactotropes.
Previous works have demonstrated the cell-specific distribution of PC1 and PC2 in several neuroendocrine and endocrine cells. These studies were related to the expression of these enzymes in pancreatic ß-cells (
The present work also provides original information about the subcellular distribution of these proteolytic enzymes. Both are present in the secretory granules of these cells, but they display other different major localization sites along the secretory pathway. PC1 is mainly observed in the Golgi apparatus and PC2 in the RER. As has been previously pointed out for the immunolabeling of PRL (
Several groups had already shown the presence of PC2 (
The presence of both PCs in PRL cells raises the question of the precursors that are proteolytically processed within these cells. The major secretory product of lactotropes is PRL, which is released into the medium without further major proteolytic processing other than the removal of the signal peptide in the RER. Some minor proteolytic products have been identified but do not correspond to potential cleavage sites by a PC (reviewed in
In conclusion, the observations reported here demonstrate the expression of PC1 and PC2 in lactotropes of the rat anterior pituitary and strengthen the role of these two prohormone convertases in the proteolytic maturation of precursors of secretory proteins within this cell type.
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Footnotes |
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1 Present address: Department of Biochemistry and Molecular Biology, LSUMC, New Orleans, LA.
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Acknowledgments |
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Supported by grants from INSERM (U 36).
We gratefully acknowledge Dr Iris Lindberg (New Orleans) for helpful discussions. We also thank Eric Etienne for the photographic work. We acknowledge the National Institute of Diabetes and Digestive, and Kidney Diseases (NIDDK) for the gift of reference rat PRL (rat PRL-RP3).
Received for publication April 28, 1997; accepted July 10, 1997.
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