Expression of enteropeptidase in differentiated enterocytes, goblet cells, and the tumor cells in human duodenum
Takahisa Imamura1 and
Yasunori Kitamoto2
1Division of Molecular Pathology, Kumamoto University Graduate School of Medical and Pharmaceutical Sciences and 2Department of Internal Medicine, Kumamoto University School of Medicine, Kumamoto 860-0811, Japan
Submitted 30 April 2003
; accepted in final form 31 July 2003
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ABSTRACT
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Enteropeptidase (EP) is a serine proteinase and activates trypsinogen to trypsin, thus playing an important role in food digestion. Nevertheless, the localization of EP is still controversial, likely due to a lack of studies using specific antibodies against EP. The aim of this study was to define cellular localization of EP in human duodenum and expression in tumor cells at the duodenal region. Immunohistochemical staining for resected tissues was performed with two antibodies against recombinant EP light and heavy chains, respectively. In situ hybridization was done with two RNA probes that include either the light or the heavy chain sequences of proEP, respectively. The two antibodies reacted with enterocytes, accentuated on the brush border, and goblet cells, with increasing intensity from the bottom of crypts to the top of villi. Paneth cells, neuroendocrine cells, Brunner's glands, lymphocytes, smooth muscle, or connective tissue did not react with the antibodies. The two RNA probes detected EP mRNA expression only in enterocytes and goblet cells. EP is produced in enterocytes and goblet cells, and the localization on the brush border of the cells is reasonable for the physiological activation of digestive enzymes. Interestingly, the antibodies reacted with tumor cells in duodenal polyps and adenocarcinoma at the duodenum but not in Brunner's gland adenoma. EP seems to be a marker of differentiated enterocytes and goblet cells, which suggests the existence of a common progenitor of these cells. Furthermore, EP may be a useful marker of tumor cells originating from these cells.
cell marker
ENTEROPEPTIDASE (EP) is a serine proteinase that is produced as a single-chain proform (proEP), sorted to apical membranes, and activated to EP (20, 27). EP cleaves off the amino-terminal activation peptide from inactive trypsinogen to produce active trypsin (6). Trypsin, in turn, activates other proenzymes, including chymotrypsinogen, procarboxypeptidases, and proelastase (1), which initiate the proteolytic digestion of proteins in the duodenum. The cDNA of human proEP is composed of 3,696 nt and contains an open reading frame of 3,057 nt. The cDNA encodes 784 amino acids of heavy chain and 235 amino acids of light chain that are homologous to other trypsinlike serine proteinases (14). After cleavage of single-chain proEP, the two chains are linked by a disulfide bond. The physiological importance of EP is indicated by severe intestinal malabsorption in congenital deficiency of this enzyme (10, 11).
The origin and cellular localization of EP have been in controversy. It appears to be agreed that EP is present on the brush border of duodenum (12, 22, 23, 26), where the enzyme activates trypsinogen. Miyoshi et al. (22) showed the localization of EP antigen in human duodenal goblet cells, although Hermon-Taylor et al. (12) and Yuan et al. (26) reported negative results with immunohistochemistry in human and in situ hybridization in mouse, respectively. Eggermont et al. (8) proposed that the Brunner's glands may be involved in the formation and secretion of EP, which, however, was denied in other reports (12, 23, 26). Most reports have shown limited localization of EP in the duodenum and upper jejunum (12, 22, 26), but Takano et al. (24) demonstrated EP antigen in goblet cells of porcine small and large intestines. These variable results may be caused partly by insufficient specificity of antibodies for EP. Taking advantage of recombinant technology, we prepared recombinant full-length proEP and its light chain and raised two antibodies specific for the heavy and light chain, respectively. To define the localization of EP, we performed immunohistochemical staining for human duodenal tissues with these antibodies and compared the results with those of in situ hybridization by using RNA probes coding the light and heavy chain sequences of proEP, respectively. Moreover, we examined the localization of EP antigen in duodenal tumor cells.
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MATERIALS AND METHODS
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Preparation of tissue sections. Tissues from surgical specimens were obtained from the Kumamoto Regional Medical Center. Written informed consent was obtained from all subjects, and the study protocol conformed to the ethical guidelines of the 1975 Helsinki Declaration. Surgically resected tissues were kept at 4°C and fixed in formalin within 1 h after the resection. After fixation for 6 h, tissue sections were embedded in paraffin through the routine procedure.
Preparation of antibodies. Polyclonal rabbit antibody anti-Lv was raised against recombinant bovine EP light chain expressed in baculovirus and purified by chromatography on protein A-agarose as described previously (27). IgG to bovine EP light chain was purified with a protein A-Sepharose column and stored at -70°C. Plasmid pHEP-full containing the full-length cDNA sequence of human EP was assembled in vector pBluescript II KS+ (Stratagene) from cDNA clones isolated previously (14). Part of the cDNA insert of pHEP-full, containing the sequence encoding the amino acids 97-1019 was cloned with the BamHI site of plasmid pET 28c(+) (Novagen, Madison, WI) to yield plasmid pET-HEP. Epicurean coli BL21(DE3) (Stratagene, La Jolla, CA) transformed with plasmid pET-HEP was grown in Luria-Bertani broth containing ampicillin, and protein expression was induced with 1 mM isopropyl-1-thio-
-D-galactopyranoside. Cells were collected by centrifugation, resuspended in 50 mM Tris · HCl (pH 8.0), 2 mM EDTA, 100 mg/ml of lysozyme, and 0.05% Triton X-100, and sonicated for lysis. The suspension was centrifuged, and inclusion bodies in the precipitate were dissolved in sample loading buffer with
-mercaptoethanol, boiled, and submitted to SDS-polyacrylamide gel electrophoresis with 10% gel. Bands were visualized with 0.1 M KCl at 4°C. and the band with molecular weight around 118 kDa was cut out, homogenized, and used to raise polyclonal antibodies in rabbit by standard methods. The IgG fraction of the immune serum was purified using protein A-conjugated Sepharose (Hi Trap protein A column; Amersham Pharmacia Biotech, Amersham, UK). For immunoblotting, calf EP (Biozyme Laboratories, Blaenavon, UK) was applied to SDS-polyacrylamide gel electrophoresis in the presence or absence of
-mercaptoethanol and transferred by electroblotting onto polyvinylidene difluoride membrane (Millipore, Bedford, MA). The membrane was blocked with 1% nonfat milk in 20 mM Tris · HCl (pH 7.5) and 150 mM NaCl (TBS) containing 0.1% Tween 20 (TBST) at room temperature for 60 min. The blotting membrane was incubated with anti-proEP antiserum (1:4,000) diluted in TBST containing 1.5% nonfat milk at 4°C overnight. The membrane was washed three times with TBST and incubated with peroxidase-conjugated antirabbit goat IgG 0.125 µg/ml in TBST containing 1.5% milk at room temperature for 1 h. After three washes with TBST, bound antibody was detected with the enhanced chemiluminescence detection system (ECL; Amersham Pharmacia Biotec). The polyclonal antibody to recombinant proEP reacted with the heavy chain of bovine EP (Biozyme Laboratories, San Diego, CA) with a molecular mass of 127 kDa but did not react with the light chain (Fig. 1).

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Fig. 1. Immunoreactivity of polyclonal antibody raised against recombinant human proenteropeptidase (proEP). Bovine EP was applied to a SDS-polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane. The antibody reacted with EP and EP heavy chain but not with EP light chain. A and B: SDS-PAGE stained with Coomassie blue; C and D: immunoblot visualized by chemiluminescence. A and C, nonreduced condition; B and D, reduced condition.
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Immunohistochemical staining. Immunohistochemical staining was performed on paraffin-embedded tissue sections (2-µm thickness) of formalin-fixed tissues. After deparaffinizing and hydration through xylene and graded alcohol series, sections were autoclaved in 50 mM citrate buffer (pH 6.0) for 10 min. The sections were then washed in 10 mM Tris · HCl buffer (pH 7.3) containing 150 mM NaCl (TBS) for 5 min (TBS wash) and incubated in 0.3% H2O2 in methanol to quench endogenous peroxidase activity. After TBS wash, sections were incubated in 3% goat serum for 30 min to eliminate nonspecific binding of antibodies. After incubation with an antibody (6 µg/ml TBS) at room temperature for 1 h, sections were stained by using a LSAB kit (DAKO, Carpenteria, CA) and diaminobenzidine tetrahydrochloride according to the manufacturer's instructions. These sections were washed in distilled water, lightly counterstained with Mayer's hematoxylin, and mounted in Aquatex (Merck, Darmstadt, Germany). Each anti-EP antibody was treated with an excess of purified bovine EP (SERVA Elecrophoresis, Heidelberg, Germany) (10 µg EP for 6 µg antibody), and the supernatants were used for the control staining. A pair of mirror sections were stained with anti-EP light chain antibody and anti-chromogranin antibody (DAKO) (100x dilution), respectively.
In situ hybridization. Human enterokinase (HEK)-27 cDNA clone containing nt 1-2362 and HEK-1 cDNA clone containing nt 2454-3668 (14) of the human EP gene were used to prepare probes for in situ hybridization. HEK-27 included 40 nt of the 5' noncoding region and 99% of the heavy chain sequence, and HEK-1 included 92% of the light chain sequence and 599 nt of the 3' noncoding region. HEK-1, subcloned in pBluescript M13+ (Stratagene), was linealized at the BamHI (antisense template) or AccI site (sense template). HEK-27, subcloned in pBluescript II KS+ (Stratagene), was linealized at the BamHI (antisense template) or HindIII site (sense template). Each template was used to transcribe in vitro single-stranded RNA with T3 RNA polymerase (antisense probe) or T7 RNA polymerase (sense probe). Transcripts were alkaline hydrolyzed, and RNA fragments of
800 nt were used as probes, which were labeled with digoxigenin.
The hybridization procedures used in this study were essentially the same as reported previously (16). Briefly, deparaffinized sections were treated with 0.2 N hydrochloric acid for 20 min at room temperature, washed with diethyl pyrocarbonate water three times for 5 min each, digested with proteinasEP (5-10 µg/ml in phosphate-buffered saline, PBS) for 8 min at 37°C, washed three times with PBS for 5 min each, and postfixed with 4% paraformaldehyde for 5 min at room temperature. After being washed with PBS for 5 min, treated sections were immersed in 0.2% glycine in PBS twice for 15 min each at room temperature, washed with PBS three times for 5 min each, and processed for in situ hybridization at 42°C (HEK-27) or 50°C (HEK-1) overnight in a mixture containing the digoxigenin-labeled probe, 50% formamide, 10 mM Tris · HCl buffer (pH 8.0), yeast tRNA (200 µg/ml), 1x Denhardt's solution, 10% dextran sulfate, 600 mM NaCl, 1 mM EDTA, and 0.25% SDS. After hybridization, the sections were washed once with 50% formamide in 2x SSC for 60 min at 45°C and washed twice with 10 mM Tris · HCl (pH 8.0) and 500 mM NaCl for 5 min. They were treated with RNase A (20 µg/ml) at 37°C for 30 min and washed with 50% formamide in 2x SSC for 60 min at 45°C, 50% formamide in 1x SSC for 60 min at 45°C, 50% formamide in 1x SSC for 30 min at room temperature, and 100 mM Tris · HCl (pH 7.5) and 150 mM NaCl three times for 5 min each. After sections were treated with 1.5% blocking reagent at room temperature for 60 min, they were reacted with alkaline phosphatase-labeled antidigoxigenin antibody for 30 min at 37°C. Afterwards, sections were washed four times with 100 mM Tris · HCl (pH 7.5) and 150 mM NaCl for 5 min each and then washed once with 100 mM Tris · HCl (pH 9.5), 100 mM NaCl, and 50 mM MgCl2 for 5 min. Sections were colored with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate with 2 mM levamizol. The reaction was stopped with 10 mM Tris · HCl (pH 8.0) and 1 mM EDTA, and the sections were counterstained with methyl green. Sections pretreated with RNase as another negative control were digested with RNase A (100 µg/ml) for 60 min at 37°C before hybridization procedures were started.
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RESULTS
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EP antigen expression in duodenum. To investigate EP localization, we performed immunohistochemical staining for human duodenal tissue using anti-EP light chain antibody. The EP antigen was positive in epithelial cells of the villi but negative in the Brunner's glands, lymphocytes, smooth muscle, and connective tissue (Fig. 2A). Enterocytes and goblet cells were stained with the antibody, and the intensity of the staining increased from the bottom of crypt to the top of villi (Fig. 2B). The antibody that recognizes EP heavy chain also stained only enterocytes and goblet cells in villi (Fig. 2, C and D), as did anti-light chain antibody (Fig. 2, A and B). Particularly, the brush border of enterocytes in the upper part of villi was strongly positive (Fig. 2E). The positive staining for these cells was abrogated when these antibodies were treated with an excess of purified EP (Fig. 2F). Paneth cells, which localize at the bottom of the crypts and have the numerous granules at the cell apex, were EP negative (Fig. 2G). The mirror sections stained with anti-chromogranin A antibody, which reacts with neuroendocrine cells (9), and anti-EP antibody revealed that neuroendocrine cells, present mostly around the base of crypts and less along the villi as shown previously (9), were EP negative (Fig. 2, H and I). These results indicate that EP is present in enterocytes and goblet cells but absent in the other types of epithelial cells and submucosal cells in human duodenum.

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Fig. 2. Immunohistochemical staining of the human duodenal tissue with anti-EP antibodies. A, B, G, and I: anti-EP light chain antibody. C-E: anti-EP heavy chain antibody. F: anti-EP heavy chain antibody treated with purified EP. H: anti-chromogranin antibody. H and I are mirror sections. Magnification: x40 (A, C, F); x100 (B, D); x400 (E); x330 (G); x90 (H, I). Asterisks indicate Brunner's glands. Arrows in E and G indicate brush border membrane and Paneth cells, respectively; arrowheads in H indicate neuroendocrine cells.
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EP mRNA expression in duodenum. To determine whether EP antigen-positive cells synthesize EP, we performed in situ hybridization for duodenal tissue targeting light chain or heavy chain sequences of EP mRNA. The antisense RNA probe of HEK-1, which includes the light chain sequence and the 3' noncoding region, reacted to epithelial cells of villi but not cells of Brunner's glands (Fig. 3A). Both enterocytes and goblet cells were stained with the probe (Fig. 3B). No positive staining was shown with a sense probe (Fig. 3C). When the tissue section was pretreated with RNase and hybridized with the antisense probe, no positive staining was shown (Fig. 3D). Both enterocytes and goblet cells were also positive for the antisense RNA probe of HEK-27, including the heavy chain sequence and the 5' non-reading region. The staining intensity increased from the crypt to the top of villi (Fig. 3E), as shown by immunohistochemical staining (Fig. 2B). No cell showed positive signals when hybridized with sense RNA probes of HEK-27 (Fig. 3F). No cells showed positive staining when the tissue section was pretreated with RNase and hybridized with the HEK-27 sense probe (data not shown). Consistent with the results of immunohistochemical staining in Fig. 2G, Paneth cells or neuroendocrine cells were negative for EP mRNA as studied by hybridization using HEK-1 and HEK-27 probes (data not shown). From these results, it appears that only enterocytes and goblet cells synthesize EP.

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Fig. 3. In situ hybridization of the human duodenal tissue with proEP RNA probes. A and B: sections hybridized with human enterokinase (HEK)-1 antisense probe. C: section hybridized with HEK-1 sense probe. D: section pretreated with RNase and hybridized with HEK-1 antisense probe. E: HEK-27 antisense probe. F: HEK-27 sense probe. Magnification: x40 (A); x100 (B-E); x200 (F).
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EP antigen expression of primary tumor cells in duodenum. To determine whether tumor cells express EP and whether EP expression can be used as a marker for the duodenal cancer, we performed immunohistochemical staining on primary tumors of duodenum including the ampulla of Vater for EP by using anti-light or -heavy chain antibodies. In benign polyp tissues composed of hyperplastic goblet cells and residual enterocytes, the cytoplasm of both cell types stained positively with the anti-light chain antibody (Fig. 4A). In enterocytes, the brush border membrane and the apical side of the cytoplasm were strongly stained. Even in hyperplastic goblet cells, EP antigen was present on the luminal surface and in the basilar cytoplasm but not in the mucous vacuoles. In adenocarcinoma, the cytoplasm of tumor cells was positive with the anti-EP light chain antibody (Fig. 4B) and the anti-EP heavy chain antibody (data not shown), which indicated that the adenocarcinoma cells express EP. The malignant cells were less polarized, and the tendency for stronger staining toward the luminal side of cells, which was observed in normal enterocytes and benign polyps, was no longer observed. Nonimmune IgG did not stain any tumor cells (data not shown). Brunner's gland adenoma cells were EP negative (Fig. 4C), as were normal Brunner's gland cells (Fig. 2A). These results indicate that tumor cells originating from duodenal enterocytes and goblet cells can express EP antigen irrespective of whether the cells are benign or malignant.

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Fig. 4. Immunohistochemical staining of duodenal tumors with the antibody to EP light chain. A: papillary adenoma. Arrows indicate enterocytes, and arrowheads indicate goblet cells. B: well-differentiated adenocarcinoma. C: Brunner's gland adenoma. Arrows indicate adenoma cells. Magnification: x200 (A, B); x90 (C).
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DISCUSSION
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EP is composed of a light chain, which is a trypsinlike catalytic subunit, and a heavy chain, which has a unique domain structure (15) and determines substrate specificity (20). Single-chain proEP is synthesized, targeted to apical cell membranes, and activated by cleavage to EP (20). We have investigated EP localization in human duodenal tissue using a strategy based on this subunit structure and cDNA sequence. We prepared recombinant proteins and raised antibodies recognizing the light and heavy chain, respectively. Both antibodies recognized proEP in cytoplasm as well as active EP on cell membranes (27). To address discrepancies among previous reports concerning EP expression (12, 22), we used these highly EP-specific antibodies in the present immunohistochemical studies and performed in situ hybridization with two probes specific for proEP mRNA.
The localization of EP on the brush border of duodenal epithelial cells (Fig. 2E) is relevant to the physiological activation of trypsinogen in the duodenum. The polarity of distribution of EP in the enterocyte coincides well with sorting of proEP to apical membrane of cells (28). The absence of EP antigen in Brunner's glands (Fig. 2, A and C) is consistent with reports that no EP activity is found in these gland (19, 23). Together with the localization of Brunner's glands remote from intestinal mucosa under a layer of muscularis mucosae, this result supports the deviation of enterocytes and epithelial cells of Brunner's glands from different cell lineages. EP antigen also was absent in Paneth cells and neuroendocrine cells (Fig. 2, G-I), which is consistent with the result of in situ hybridization showing absence of mRNA in the mouse tissue (26). The extensive agreement between the localization of EP antigen and mRNA suggests that EP is produced only in enterocytes and goblet cells.
Both EP antigen (Fig. 2B) and mRNA (Fig. 3E) gradually increased from the crypts of Lieberkühn to the top of villi. This tendency is in accordance with reports that EP activity is absent in crypts (23) but significant in villous enterocytes and maximal in the upper half of the villi (19). Intestinal stem cells in the bottom of the crypt differentiate into four cell types in epithelial flow. Stem cells give rise to two types of long-lived daughters; one (Co) is committed to produce column-shaped enterocytes, and the other (Mo) is thought to yield mucus-producing goblet cells (3). Neuroendocrine cells and Paneth cells are thought to arise from Mo cells in this model (4, 21). EP expression in enterocytes and goblet cells but not in neuroendocrine cells or Paneth cells may suggest the existence of the EP-positive progenitor cell that is derived from stem cells and yields both Co and Mo cells. Because EP is expressed in differentiated but not in undifferentiated enterocytes, it can be a marker of differentiated enterocytes. The presence of EP in tumor cells of enterocyte origin (Fig. 4) indicates that enterocytes continue to express EP even after transformation. Therefore, EP also could be useful to diagnose tumor cells of enterocyte origin.
It is reported that proteinase-activated receptor 2 is present at the apical and basolateral membrane of enterocytes and that activation of this receptor by trypsin stimulates enterocytes to secrete eicosanoids (17), which act locally in the intestinal wall to regulate epithelial growth (7). Therefore, EP localization on the luminal surface of the duodenal villi may contribute to enterocyte growth by generating active trypsin on the cell surface. Recently, it was reported that trypsinogen is expressed by various human epithelial cells (18) and is also secreted by human tumor cells including gastric (13) and colon cancer cells (2). Trypsinogen production was associated with metastatic phenotypes of these human cancer cells (13, 25). Human colon cancer cell proliferation was initiated by trypsin through activation of the protease-activated receptor-2 (PAR-2) (5). EP expression in tumor cells (Fig. 4) suggests that the tumor EP activates the tumor cell-derived trypsinogen, producing trypsin, which in turn activates PAR-2 on the tumor cells, leading to promotion of tumor cell growth and invasion.
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DISCLOSURES
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This work was supported by The Japanese Ministry of Education and Science Grants 11670219 (to T. Imamura) and 12670984 (to Y. Kitamoto).
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ACKNOWLEDGMENTS
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We thank Tatsuko Kubo for technical and secretarial assistance. We thank Dr. J. Evan Sadler for critical reading of the manuscript.
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FOOTNOTES
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Address for reprint requests and other correspondence: Y. Kitamoto, The Third Dept. of Internal Medicine, Kumamoto Univ. School of Medicine, 2-2-1 Honjo, Kumamoto 860-0811, Japan (E-mail: yasunori{at}kaiju.medic.kumamoto-u.ac.jp).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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Copyright © 2003 by the American Physiological Society.