ARTICLE |
Correspondence to: Merel C. Strik, Dept. of Clinical Chemistry, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands. E-mail: m.strik@vumc.nl
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Summary |
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Ovalbumin-like serine protease inhibitors are mainly localized intracellularly and their in vivo functions are largely unknown. To elucidate their physiological role(s), we studied the expression of one of these inhibitors, protease inhibitor 8 (PI-8), in normal human tissues by immunohistochemistry using a PI-8-specific monoclonal antibody. PI-8 was strongly expressed in the nuclei of squamous epithelium of mouth, pharynx, esophagus, and epidermis, and by the epithelial layer of skin appendages, particularly by more differentiated epithelial cells. PI-8 was also expressed by monocytes and by neuroendocrine cells in the pituitary gland, pancreas, and digestive tract. Monocytes showed nuclear and cytoplasmic localization of PI-8, whereas neuroendocrine cells showed only cytoplasmic staining. In vitro nuclear localization of PI-8 was confirmed by confocal analysis using serpin-transfected HeLa cells. Furthermore, mutation of the P1 residue did not affect the subcellular distribution pattern of PI-8, indicating that its nuclear localization is independent of the interaction with its target protease. We conclude that PI-8 has a unique distribution pattern in human tissues compared to the distribution patterns of other intracellular serpins. Additional studies must be performed to elucidate its physiological role.
(J Histochem Cytochem 50:14431453, 2002)
Key Words: serpin, human, nucleus, PI-8, immunohistochemistry
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Introduction |
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Serine protease inhibitors (serpins) are a large superfamily of structurally related proteins that occur in viruses, insects, plants and higher organisms, but not in bacteria or yeast (
Recently, a subfamily of serpins, of which chicken ovalbumin is the archetype, with substantial higher homology (around 50%) has been identified (
The physiological role of intracellular serpins remains largely unknown, in part because their cognate substrates (serine proteases) are not yet identified. Although in vitro studies have been performed to identify target proteases, most of the proteases identified reside extracellularly and are therefore probably not the physiological target proteases in vivo. In addition, knowledge about the site of expression of these serpins in vivo is of importance to understand their function. Although ovalbumin-like serpins are widely expressed among human tissues, each has its own restricted expression pattern. For example, the PI-8-related serpin PI-9 is mainly expressed by dendritic cells, endothelial cells, cytotoxic lymphocytes, and cells of immune-privileged sites (
PI-8 is a 45-kD serpin with arginine at the P1 position in its RSL, indicating that it probably inhibits trypsin-like proteases. PI-8 inhibits trypsin, thrombin, factor Xa, subtilisin A, furin, and also chymotrypsin in vitro (
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Materials and Methods |
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Materials and Antibodies
Dulbecco's modified Eagle's medium (DMEM), RPMI 1640, and Iscove's modified Dulbecco's medium (IMDM) were obtained from Bio-Whittaker Europe (Verviers, Belgium). Fetal bovine serum (FBS) was purchased from Life Technologies (Rockville, MD). All primers were synthesized by Eurogentec (Brussels, Belgium). Qiaex II Gel Extraction Kit and Qiagen Plasmid Maxi Kit were obtained from Qiagen (Hilden, Germany). JM109 high-efficiency competent cells were obtained from Promega (Madison, WI). The ABI prism sequence kit Thermo Sequenase DNA sequencing kit was purchased from Amersham (Arlington Heights, IL). The following reagents were obtained from DAKO (Glostrup, Denmark): normal rabbit serum, normal swine serum, rabbit anti-human glucagon antibody, mouse anti-human thyroid-stimulating hormone (TSH) antibody, mouse anti-human chromogranin antibody, biotinylated rabbit anti-mouse F(ab)2 Ig, biotinylated swine anti-rabbit F(ab)2, HRP-conjugated rabbit anti-mouse Ig, HRP-conjugated swine anti-rabbit Ig, FITC-conjugated rabbit anti-mouse Ig, avidinbiotinHRP complex (sABC), biotinylated tyramine (BT), and streptavidinFITC. Biotin-labeled goat anti-mouse IgG1 and HRP-labeled goat anti-mouse IgG2a Abs were obtained from Southern Biotechnology Associates (Birmingham, AL) and tyraminerhodamine from DuPont Pharmaceuticals (Wilmington, DE). FuGENE 6 transfection reagent was obtained from Roche Molecular Biochemicals (Indianapolis, IN). 4-(2-aminoethyl)-benzenesulfonylfluoride-HCl was obtained from AG Scientific (San Diego, CA), propidium iodide from Alexis Biochemicals (Lausanne, Switzerland), and Nonidet P-40 and poly-L-lysine from Sigma (St Louis, MO). The Micro BCA Protein Assay was obtained from Pierce (Rockford, IL).
The anti-PI-9 monoclonal antibodies MAb PI-9-17 and MAb PI-9-1 were produced as described previously (
Plasmids and Cloning
The constructs PI-6-pcDNA3, PI-8-pcDNA3, and PI-9-pcDNA3.1, comprising the cDNA sequence coding for the full-length proteins, were prepared as previously described (
The point mutation of the P1 residue (Arg321Ala321) in the RSL of PI-8 was induced by the PCR method using the PI-8-pcDNA3 plasmid as a template. The PI-8-P1(argala) construct was amplified with a Kozak sequence (GCC ACC) in front of the ATG and cloned as a BamHI/XhoI fragment into PCR-3 (Invitrogen) plasmid and sequenced on both strands to confirm the mutation. PI-8 without the first ATG (amino acids 2374) was cloned into a modified PCR-3 vector containing a FLAG sequence as a SalI/XhoI fragment. From the N-terminal side the amino acid sequence is M(DYKDDDDK)EFCRYPSHWRPLDDDL with the FLAG sequence between brackets and the PI-8 sequence in bold.
Cell Culture and Transfection
Cos-1 cells (CRL-1650, American Type Culture Collection, Manassas, VA) were cultured in IMDM supplemented with 5% (v/v) heat-inactivated FBS, 2 mM L-glutamine, and penicillin/streptomycin (fc. 50 IU/ml and 50 µg/ml, respectively). HeLa cells (CCL-2; ATCC) were cultured in RPMI 1640 medium supplemented with 5% (v/v) heat-inactivated FBS, 2 mM L-glutamine, and antibiotics. All cells were maintained at 5% CO2/95% air in a humidified incubator at 37C.
Cos-1 or HeLa cells were grown in 25-cm2 culture flasks or on sterile glass coverslips in 6-well plates for 24 hr. Medium was refreshed just before transfection with plasmid DNA. Cells were transfected with either PI-6-pcDNA3, PI-8-pcDNA3, PI-9-pcDNA3.1, PAI-2-pCI, PI-8-P1(argala)-PCR3, or an empty control plasmid. Transfection was performed with FuGENE 6 transfection reagent according to the manufacturer's instructions. FuGENE 6 transfection reagent (microliters) to plasmid DNA (micrograms) was used in a ratio of 3:2 and subsequently cultured for at least 48 hr. The transfected cells on coverslips were further analyzed by immunofluorescence staining (see below). For production of cell lysates, cells in the plates were washed twice with PBS and lysed by adding lysis buffer [PBS with 0.2% (w/v) Nonidet P-40] directly in the culture plate. Cells were incubated for 15 min on ice, after which the lysate was harvested and centrifuged for 10 min at 3000 rpm to remove cell debris and DNA. The supernatant (cell lysate) was stored at -80C until further use.
Immunohisto/cytochemistry and Immunofluorescence Staining
Slides with sections of formalin-fixed, paraffin-embedded normal human tissues were obtained from the tissue bank of the Department of Pathology, VU University Medical Center (Amsterdam, The Netherlands). All tissues were sampled from surgical specimens within 2 hr after resection. Formalin-fixed [10% (v/v) for 18 hr], paraffin-embedded tissue was used. Sections (3 µm thick) were mounted on poly-L-lysine-coated tissue slides and deparaffinized. Cytospins of serpin-transfected cells were fixed in 10% formalin for 10 min. Endogenous peroxidase activity was blocked by incubation in 0.3% (v/v) H2O2 in methanol for 30 min. Unless stated otherwise, tissue sections and cytospins were subjected to antigen retrieval by boiling in 10 mM, pH 6.0, sodium-citrate buffer for 10 min in a microwave oven. All antibodies and normal serum were diluted in PBS containing 1% (w/v) bovine serum albumin (BSA). Tissue sections were pre-incubated for 10 min with normal rabbit or swine serum, followed by incubation for 1 hr with purified MAb anti-PI-8k (at 1.4 µg/ml). To identify specific cell populations, sequential sections of several tissues were incubated for 1 hr with the appropriate Abs against various cell markers. Fig 2 shows staining with the following Abs: rabbit anti-human glucagon (1:100), mouse anti-human TSH (1:100), and mouse anti-human chromogranin (1:100). After washing with PBS, slides were incubated with a biotin-conjugated secondary antibody (rabbit anti-mouse F(ab')2 Ig, 1:500 dilution; swine anti rabbit F(ab')2, 1:300 dilution) for 30 min. Slides were incubated with streptavidinbiotinHRP complex (sABC; 1:1000 dilution) for 1 hr, followed by incubation with biotinylated tyramine (BT) for 10 min. After a second incubation with sABC (1:200), PI-8 or cell markers were visualized with 3-amino-9-ethylcarbazole (AEC) or 3,3'-diaminobenzidine (DAB; 0.1 mg/ml, 0.02% H2O2). Cytospins were incubated with AEC directly after the first incubation with sABC. Slides were counterstained with hematoxylin and mounted with Depex. Negative control slides were stained with mouse IgG of the appropriate subclass.
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For immunofluorescence staining of coverslips containing transfected Cos-1 or HeLa cells, slides were washed twice in PBS, fixed in 10% formalin for 10 min at RT, and washed again in PBS. Cells were then permeabilized in PBS with 0.5% (w/v) Nonidet P-40 for 10 min at RT. Incubation for 5 min in PBS with 0.75% (w/v) glycin, followed by two incubations for 10 min in PBS with 0.5% BSA, were performed as blocking steps before the immune fluorescence labeling. All antibodies were diluted in PBS with 0.5% BSA. Fixed cells were stained for PI-8 with purified MAb anti-PI-8k (at 1.1 µg/ml). PI-9 was detected using MAb PI-9-17 at a concentration of 2 µg/ml. On washing with PBS with 0.5% BSA, the cells were incubated in the dark with the secondary antibody, FITC-labeled rabbit anti-mouse F(ab')2 Ig diluted 1:500. Cells were counterstained with propidium iodide (20 µg/ml), diluted 1:100 in PBS with 0.5% BSA for 5 min and mounted in Vectashield (Vector Laboratories; Burlingame, CA). Images were recorded using a Zeiss LSM-510 confocal laser scanning microscope equipped with argon and helium/neon lasers. Excitation was at 488 nm for FITC and 568 nm for propidium iodide.
Western Blotting
About 20 µg of tissue section lysate protein or 10 µl of cell lysate (4.105 cells) from serpin transfected Cos-1 or HeLa cells was resolved by electrophoresis on a 10% (w/v) SDS-polyacrylamide gel under reducing conditions. After electrophoresis, proteins were transferred to nitrocellulose membranes by electrophoretic blotting. After transfer, the membranes were blocked for 1 hr in blocking buffer [5% (w/v) skim milk powder, 0.5% (w/v) BSA, 0.1% (w/v) Tween- 20 in PBS]. Membranes were then incubated overnight at 4C with MAb anti-PI-8k at a concentration of 0.6 µg/ml in blocking buffer and washed in PBS containing 0.1% (w/v) Tween. The membranes were incubated for 1 hr with HRP-conjugated rabbit anti-mouse Ig (1:1000 dilution in blocking buffer), followed by another washing step. Bound Abs were visualized with a chemiluminescence development reagent (ECL system; Amersham) according to the manufacturer's instructions.
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Results |
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Characterization of MAb Anti-PI-8k Directed Against Human PI-8
A panel of MAbs was obtained from a fusion experiment of a mouse immunized with recombinant human PI-8 produced in a Pichia pastoris expression system (
On the basis of these data, MAb anti-PI-8k was considered to be specific for PI-8 and was used for further studies.
PI-8 Is Mainly Expressed in Nuclei of Epithelial Cells
Analysis of the expression of PI-8 in normal human tissues by immunohistochemistry (Fig 2) showed that PI-8 was prominently expressed in the nuclei of squamous epithelium. Fig 2a shows a tonsil lined with squamous epithelium in which the nuclei express PI-8. The lymphoid tissue lying beneath the epithelium (Fig 2a, asterisk) does not contain PI-8. A higher magnification of the squamous epithelial layer shown in Fig 2a is depicted in Fig 2b. The basal cells do not express PI-8 (Fig 2b, arrow), while PI-8 expression in the nuclei of keratinocytes strongly increases during epithelial differentiation (Fig 2b and Fig 2d, arrowheads). PI-8 protein occurs mainly in the nuclei, whereas the cytoplasm of these cells does not contain measurable amounts of the serpin. The same pattern was observed in squamous epithelia from other sites, such as the esophagus (Fig 2c) and skin (Fig 2d). The nuclei of epithelial cells present in skin appendages, such as hair follicles (Fig 2e), also expressed PI-8. Interestingly, nuclei from epithelial cells present in the Hassall's corpuscles in the thymus were also positive for PI-8 (results not shown).
The other non-squamous epithelial tissue types showed a more heterogeneous pattern. Epithelium from colon (Fig 2g), lung (Fig 2k), and exocrine pancreas (Fig 2q, asterisk), salivary glands (except for the ducts), endometrium, prostate, and breast (not shown) were in general negative or sporadically positive for PI-8. In contrast, the epithelium of the antral part of the stomach (Fig 2f), the small intestine, tubes, and endocervix (not shown) showed moderate to strong nuclear staining of PI-8, whereas the cytoplasm of these cells hardly contained the serpin. In most other organs tested, such as kidney (Fig 2i), heart (Fig 2j), brain (Fig 2m), placenta (Fig 2n), and liver (not shown), the epithelial and/or mesenchymal structures did not express PI-8. As an internal positive control, PI-8-positive monocytes (see below) were detected in all cases.
PI-8 Expression by Monocytes and Neuroendocrine Cells
Monocytes detected in the blood vessels of most organs showed strong PI-8 expression. Fig 2n shows a high magnification of a monocyte (arrow) in which cytoplasmic as well as nuclear staining is observed. PI-8-positive monocytes were used as a positive internal control for the immunohistochemical staining in PI-8-negative organs (Fig 2n, arrow). Although alveolar macrophages in the lung showed weak staining (Fig 2k, arrow), other macrophage or dendritic subsets present in the lymphoid (follicular dendritic cells, sinus macrophages) or other organs (Kupffer cells in the liver) did not express PI-8 (not shown). Cells from the lymphoid system such as T-, B-, and plasma cells, did not contain detectable amounts of PI-8, as can be observed in Fig 2a and Fig 2l in which the lymphoid tissue from the tonsil or spleen (asterisk) is shown, respectively. Neutrophilic granulocytes were mainly PI-8-negative, although sometimes weak staining was observed. Endothelial cells were negative for PI-8, whereas mesothelial cells were positive (not shown).
Interestingly, high PI-8 protein levels were detected in certain neuroendocrine cells of the pituitary gland (Fig 2o), endocrine pancreas (islets of Langerhans, Fig 2q), paraganglia (not shown), and gastrointestinal tract (Fig 2g). In the pituitary gland, only the TSH-producing cells expressed PI-8 (Fig 2o), as confirmed by sequential staining for TSH (Fig 2p). The other hormone-producing cells of the pituitary gland were mainly PI-8-negative. In the islets of Langerhans of the pancreas, the cells arranged around the periphery expressed PI-8 (Fig 2q). Sequential staining identified these cells as the glucagon-producing or -cells (Fig 2r), whereas the insulin-producing or ß-cells in the central region of the islet were PI-8-negative (not shown). Neuroendocrine cells in the gastrointestinal tract also contained PI-8. Fig 2g shows several scattered PI-8-positive cells in the crypts of the colon mucosa (arrowhead). The cells were lying between the PI-8-negative epithelial cells, and sequential staining for the neuroendocrine marker chromogranin (Fig 2h, arrowhead) showed that these cells belong to the neuroendocrine system. The parathyroid gland and the adrenal gland showed no PI-8 staining (results not shown). In contrast to the nuclear expression in the epithelial cells, PI-8 appears to be expressed mainly in the cytoplasm of the neuroendocrine cells, as clearly shown in Fig 2o. As negative control, all tissues were also stained using an isotype-matched irrelevant monoclonal antibody that showed no background staining (results not shown).
Western Blotting Analysis of Tissues Expressing PI-8
The distribution pattern of PI-8 among the various tissues as seen by immunohistochemistry, as well as the identity of the protein, was broadly confirmed by Western blotting (Fig 3). Consistent with the immunohistochemical data, no or hardly any PI-8 was detected in brain, heart, and renal tissues. The endocrine (pituitary gland, pancreas) and epithelial tissues were positive for PI-8. The weak expression in the pancreas was probably due to the low percentage of -cells in the pancreatic tissue. The spleen, liver, and lung also showed strong staining, presumably due to presence of high amounts of monocytes and alveolar macrophages, respectively. The PI-8 protein detected has the same Mr (45 kD) as that of the positive control consisting of HeLa cells transfected with PI-8/pcDNA3 plasmid (arrow) and represents the uncomplexed, uncleaved PI-8 protein. Interestingly, in the pituitary gland and liver there was also a higher band of 60 kD (arrowhead), and in the oropharyngeal mucosa yet another band, with an Mr of approximately 70 kD, was observed. These HMW bands may represent SDS-resistant complexes of PI-8 covalently linked to as yet unidentified target serine proteases.
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Subcellular Distribution of PI-8 In Vitro
During immunohistochemical survey of normal human tissues, PI-8 was observed in the nuclei of epithelial cells and in both the nuclei and cytoplasm of monocytes. However, no nuclear localization was observed in neuroendocrine cells.
To confirm the subcellular distribution in vitro, we analyzed the presence of PI-8 in the human epithelial cell line HeLa by using MAb anti-PI-8k in confocal microscopy. Western blotting analysis showed low levels of endogenous PI-8 in HeLa cells (Fig 3), which could not be detected by confocal microscopy analysis (Fig 4m, right). Therefore, we analyzed HeLa cells transiently transfected with the full-length PI-8 gene. Confocal microscopic analysis using MAb anti-PI-8k showed a strong nuclear staining of PI-8 in these transfected cells (Fig 4m). Interestingly, within the strong PI-8 positive nuclei, the nucleoli consistently showed a lower signal for PI-8 (Fig 4k4m and Fig 4q). As a control, HeLa cells were also transfected with a FLAG-tagged PI-8 construct, after which PI-8 was visualized using an MAb directed against the FLAG sequence. Using this approach, a similar subcellular localization of PI-8 was found, i.e., nuclear localization with lower amounts in the nucleoli (results not shown). If the primary antibody (anti-PI-8k) was omitted, no fluorescent labeling was observed, excluding nonspecific labeling with the FITC-conjugated secondary antibody (not shown). PI-8 was not or only faintly present in the cytoplasm (Fig 4). Cytoplasmic localization was mainly seen in cells with the highest PI-8 expression levels (compare cell at the left with the cell at the right in Fig 4m) and is probably due to overexpression of the protein. These in vitro data therefore support the in vivo observation that PI-8 is transported to the nucleus in epithelial cells.
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The Nuclear Localization Is Independent of the RSLP1 Residue
The presence of PI-8 in the nucleus suggests that either PI-8 itself contains a nuclear localization signal (NLS) or that PI-8 interacts with another protein in the cytoplasm that contains the appropriate signal. However, based on primary amino acid composition, PI-8, in contrast to PI-10 (
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Discussion |
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The (patho)physiological role for intracellular serpins is far from clear. Identification of the tissue or cell types expressing these serpins in vivo will provide clues to the processes regulated by these inhibitors and may help to identify their physiological target proteases. In the present study we investigated the distribution of the intracellular serpin PI-8 in normal human tissues and performed in vitro experiments to verify key findings of these studies. Although a previous study using Northern blotting analysis on human tissues showed a widespread distribution of PI-8 mRNA (
Intracellular serpins show substantial higher structural homology (about 50%) than other members of the serpin superfamily. As a consequence, antibodies raised against one of these serpins may crossreact with the other serpins. The MAb anti-PI-8k, raised against human PI-8 and used in this study, was checked for crossreactivity against the most homologous family members of PI-8, which are PI-6 and PI-9, sharing 68% and 63% amino acid identity with PI-8, respectively (
In this study we have found PI-8 expression in the nucleus of differentiated keratinocytes, whereas basal epithelial cells were PI-8 negative. These results suggest that PI-8 is related to the differentiation of these cells. The notion that intracellular serpins are involved in the cellular differentiation of keratinocytes is supported by the finding that other intracellular serpins are also expressed by differentiating epithelial cells. Epithelial cells from the epidermis also express the serpin PI-6 and, like PI-8, protein levels of PI-6 increase upon differentiation. (
Of all cells of hematopoietic origin, PI-8 was expressed only by monocytes, although sporadic staining of granulocytes was observed. PI-6, PI-10, PAI-2, and monocyte neutrophil elastase inhibitor are all expressed at different stages of monocyte differentiation and activation. (
Surprisingly, PI-8 was expressed selectively by certain cells from the neuroendocrine system, such as TSH-producing cells in the pituitary gland, glucagon-producing -cells in the pancreas, and neuroendocrine cells present in the gastrointestinal tract. Recently, a novel intracellular serpin, endopin 1, has been identified in the neurosecretory vesicles of chromaffin cells from the adrenal medulla. Endopin regulates the chromaffin granule prohormone thiol protease, which is involved in proenkephalin processing (
The most striking feature in the distribution of PI-8 in tissues was its predominant, if not exclusive, nuclear localization in epithelial cells and, to a lesser extent, in monocytes. This nuclear localization in tissues was confirmed in vitro by transient expression of the PI-8 gene in the human epithelial cell line HeLa. In these experiments, PI-8 was present in the cytoplasm as well, but only in cells that expressed the highest amounts of protein. Therefore, cytoplasmic localization in these cells may be a result of saturation of the nuclear routing. Until recently, intracellular serpins were considered to reside only in the cytoplasm, although some, such as PAI-2, are partially secreted. As a first exception, PI-10 was shown to have an insertion between the helices C and D, which contains an NLS (
Bird and co-workers, using cell lines, showed that various intracellular serpins (PI-6, PI-8, PI-9, and PAI-2) are localized in both the nucleus and cytoplasm. Although these serpins lack conventional NLS sequences, the nuclear localization was found to be an active process requiring an as yet unidentified non-conventional nuclear import pathway (
To what extent interaction with the cognate proteases modulates nuclear localization of intracellular serpins is not clear. The localization pattern of the P1 mutant of PI-8 was similar to that of wild-type PI-8 in the transfected HeLa cells, suggesting that such an interaction does not provide a positive signal for nuclear localization. On the other hand, by immunoblotting analysis we found, for the first time, evidence for PI-8/protease complexes in endocrine tissues (see Fig 3). Therefore, one could speculate that the interaction of a serpin with a cytoplasmic target proteinase inhibits nuclear translocation.
In conclusion, we show that the distribution of PI-8 in normal human tissues is mainly restricted to the nuclei of epithelial cells and that its expression is strongly related with the stage of cell differentiation. PI-8 is also detected in monocytes and in the cytoplasm of certain (neuro)endocrine cells. This distribution pattern of PI-8 in vivo suggests a relation of this serpin with differentiation of keratinocytes, processing of prohormones in neuroendocrine cells, and protection of monocytes against spillover of proteases from their lysosomal granules. Additional studies are required to elucidate the function of PI-8 in these processes.
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Acknowledgments |
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Supported by the Dutch Cancer Foundation (grant VU-98-1718) and the National Institutes of Health (grant HL64119).
Received for publication March 11, 2002; accepted May 29, 2002.
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