Expression of extracellular glutathione peroxidase in human and mouse gastrointestinal tract

Doris M. Tham, John C. Whitin, Kenneth K. Kim, Shirley X. Zhu, and Harvey J. Cohen

Department of Pediatrics, Stanford University School of Medicine, Palo Alto, California 94305

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Extracellular glutathione peroxidase (EGPx) is a glycosylated selenoprotein capable of reducing hydrogen peroxide, organic hydroperoxides, free fatty acid hydroperoxides, and phosphatidylcholine hydroperoxides. We found that human large intestinal explant cultures synthesize EGPx and cellular glutathione peroxidase (CGPx) and secrete EGPx. The level of EGPx mRNA expression relative to alpha -tubulin was similar throughout the mouse gastrointestinal tract. EGPx mRNA transcripts have been localized to mature absorptive epithelial cells in human and mouse large intestine. Western blot analysis of mouse intestinal protein has demonstrated the presence of EGPx protein in the small intestine, cecum, and large intestine, with the highest protein levels found in the cecum. Immunohistochemistry studies of human large intestine and mouse small and large intestine sections demonstrated the presence of EGPx protein within mature absorptive epithelial cells. In human large intestine and mouse small intestine, EGPx protein is also present in the extracellular milieu. These results suggest a role for EGPx in protection of the intestinal tract from peroxidative damage and/or in intercellular metabolism of peroxides.

antioxidant; epithelial cells; oxidative damage; peroxides; selenium

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

SELENIUM-DEPENDENT glutathione peroxidase (GPx) is an antioxidant enzyme that detoxifies hydrogen peroxide and lipid hydroperoxides using reduced glutathione and thus protects biomembranes and essential cellular components against oxidative damage (16). Extracellular GPx (EGPx) is genetically distinct from the classical cellular GPx (CGPx), membranous phospholipid hydroperoxide GPx (28), and an intestinal form of GPx (GPx-GI) (11). EGPx is capable of reducing hydrogen peroxide, organic hydroperoxides, free fatty acid hydroperoxides, and phosphatidylcholine hydroperoxides (14, 32-22). EGPx has been found in plasma, milk, amniotic fluid, exocoelomic fluid, and fluid from lavaged lung (3, 6, 7, 19). In adults, most of the blood plasma EGPx activity is derived from the kidney with additional sites of synthesis of EGPx mRNA in the lung, heart, and intestine (2, 5, 19). Elucidating the sites of synthesis and secretion of EGPx is necessary to gain better understanding of the function of this unique selenoglycoprotein. The human colon adenocarcinoma cell line Caco-2 is one of several cell lines that synthesizes and secretes EGPx (4). In addition, mouse intestine is also a site of synthesis of EGPx mRNA (19, 24). This study describes further investigation of the synthesis of EGPx in the intestine.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Animals. Adult BALB/c mice, at ~60 g, were the source of all experiments involving mouse intestine.

Human tissue. All described experiments involving human intestine were performed using nontumorous adult human intestinal tissues derived from material that was removed as a part of resection surgery.

Culturing of human intestinal explants and exposure to metabolic labeling with [35S]methionine. Human intestinal explants were cultured by modification of the method described by Autrup et al. (1). The specimens were cleansed of mesentery and cut into 5 × 5 mm pieces. Explants were placed in 24-well plastic tissue culture dishes coated with rat tail tendon collagen, with the epithelium facing the gas-medium interface and the serosa of the intestine resting on the collagen-coated dish. Explants were stabilized for 4 h in medium that contained DMEM (Mediatech, Herndon, VA) with 20% fetal bovine serum (FBS) (HyClone Laboratories, Logan, UT) supplemented with 1 × 10-7 M sodium selenite. Explants were placed in stabilizing medium within 30 min after surgical removal. The tissue was cultured at 37°C in a humidified incubator (Forma Scientific, Marietta, OH) with an environment of 7.5% CO2-92.5% air.

After stabilization, the medium was changed to methionine-deficient DMEM containing 10% dialyzed FBS for 30 min. The medium was then changed to methionine-deficient DMEM supplemented with 4 mM glutamine, 1 × 10-7 M sodium selenite, and 800 µCi/ml [35S]methionine (sp act >1,000 Ci/mmol, NEN, Boston, MA). After 4 h, the medium was supplemented to a final concentration of 0.02 mM cysteine and 0.02 mM methionine and the incubation continued overnight. After 24 h of incubation, the explants and the medium in which they were maintained were harvested and the selenoproteins were immunoprecipitated using rabbit anti-EGPx IgG and anti-CGPx IgG (see below). For each immunoprecipitation, duplicates of the explants or medium were pooled.

Immunoprecipitation of metabolically labeled selenoproteins from intestinal explants in culture and culture medium. Immunoprecipitation and analysis of immunoprecipitates were performed by a modification of the method of Avissar et al. (2). Metabolically labeled intestinal tissues were homogenized using a Tissue Tearor (Biospecs Products, Racine, WI) in 0.2% SDS, 2 mM N-ethylmaleimide (NEM), 2 mM iodoacetic acid (IAA), 4 mM phenylmethylsulfonyl fluoride (PMSF), and 4 mM EDTA in 0.1 M Tris buffered to pH 8.3. The cell extracts were centrifuged for 15 min at 2,500 rpm, and the supernatants were saved. An equal volume of a solution containing 1% Nonidet P-40, 1% deoxycholate, and 0.1 M Tris · HCl (pH 8.3) was used to wash the homogenizer and then added to the saved supernatants to bring the constituents to a final concentration of 0.1 M Tris, 0.1% SDS, 1 mM NEM, 1 mM IAA, 2 mM PMSF, 2 mM EDTA, 0.5% Nonidet P-40, and 0.5% deoxycholate. The culture medium was centrifuged, and the cell-free supernatant was supplemented to give a final composition of 1 mM IAA, 1 mM PMSF, 0.1% SDS, and 1 mM NEM. All samples were saved at -70°C until further processing.

The samples were thawed and incubated with 0.3 mg normal rabbit IgG plus 100 µl Pansorbin cells (Calbiochem, San Diego, CA). The samples were then centrifuged, and the cleared supernatants were immunoprecipitated with specific antibodies as follows. Protein A Sepharose CL-4B (100 µl of a 50% slurry) was incubated at 4°C for 2 h with rabbit anti-EGPx or anti-CGPx (0.3 mg, an amount sufficient to completely immunoprecipitate the respective GPx). Samples of cleared homogenized tissue or medium were added and mixed gently at 4°C for 90 min. The immunoprecipitates were exhaustively washed with ice-cold high-stringency buffer (15 mM Tris, 5 mM EDTA, 2.5 mM EGTA, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 120 mM NaCl, 25 mM KCl, and 1 mM PMSF), underlaid with 10% sucrose, collected by centrifugation at 400 g for 5 min at 4°C, washed with a high-salt buffer (15 mM Tris, 5 mM EDTA, 2.5 mM EGTA, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 1 M NaCl, and 1 mM PMSF), washed with a low-salt buffer (15 mM Tris and 5 mM EDTA), boiled in Laemmli electrophoresis sample buffer containing 100 mM dithiothreitol (DTT), and centrifuged for 1 min at 13,200 rpm. An aliquot was analyzed by 12.5% SDS-PAGE according to the method of Laemmli (21). After equilibration in an autoradiofluorography-enhancing solution (Amplify, Amersham, Piscataway, NJ), the gels were dried and exposed to X-ray film (Kodak X-OMAT AR, Eastman Kodak, New Haven, CT) for 2 wk. The bands corresponding to either EGPx or CGPx were quantified by densitometry (Quantity One, PDI, Huntington Station, NY).

Isolation and hybridization of total RNA. Alternating 2-cm intestinal sections from mice were dissected from the proximal duodenum to the distal colon. Sections were rinsed with PBS for mRNA isolation. Total RNA was isolated using a combination of the Ultraspec RNA isolation system (Biotecx, Houston, TX) and a modified Chomczynski and Sacchi method (27). We electrophoresed 10 µg of total RNA in 1.5% agarose-formaldehyde gels. The resolved RNA was transferred to positively charged Nylon membranes (Nytran Plus, Schleicher & Schuell, Keene, NH) with 20× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) and ultraviolet cross-linked (Stratalinker, Stratagene, La Jolla, CA). Random-primed (RadPrime labeling kit, GIBCO, Gaithersburg, MD) murine EGPx c-DNA (clone 47-A1, a kind gift of R. Maser, University of Kansas) was labeled with [alpha -32P]dCTP (sp act 3,000 Ci/mmol, DuPont NEN) to specific activities >1 × 108 cpm/µg. The blots were prehybridized and hybridized in 5× Denhardt's solution, 50% deionized formamide, 10% dextran sulfate, 250 µg/ml salmon sperm, 1% SDS, and 2× SSC at 42°C overnight. The blots were then washed at 55°C with the most stringent washing in 0.1× SSC and 1% SDS before exposure to a phosphor-imaging screen. The screens were scanned and quantified using a Bio-Rad GS-505 Molecular Imager (Hercules, CA). The blots were stripped in boiling 0.1% SDS for 1 min and checked for background before the next probe was used. The blots were also probed with CGPx (a gift from Dr. N. Imura, Kitasato University, Japan) and alpha -tubulin using probes prepared in identical fashion.

Preparation of RNA from villus-enriched mouse small intestine. To provide additional evidence for the localization of mRNA, we excised ~2 cm of proximal ileum from the mouse. Using the polished wide-open end of a glass pipette, we lightly scraped away the villus epithelium from the muscularis. RNA from the villus epithelium and muscularis samples were prepared and probed as in the above-described procedure, except glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was utilized in lieu of alpha -tubulin.

In situ hybridization. Normal human and mouse intestinal tissue were obtained and cut into pieces of ~27 mm3, fixed in 4% paraformaldehyde overnight, embedded in paraffin, and sectioned (5 µm) onto gelatin-subbed glass slides. After deparaffinization with xylene and rehydration through graded ethanol washes (100, 95, 70, 50, and 30%), sections were treated sequentially with 1 µg/ml proteinase K for 5 min, washed, and dipped in 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0) for 5 min. Sense and antisense cRNA probes were made from linearized plasmids containing EGPx and CGPx cDNA inserts by in vitro transcription in the presence of biotinylated nucleotides (NEN). Before hybridization, limited alkaline hydrolysis of RNA probes was performed to reduce transcript length to 0.1-0.3 kb (44 mM NaHCO3 and 66 mM Na2CO3, pH 10.2, for 25 min at 60°C). Sections were hybridized overnight at 50°C in a solution containing 50% formamide, 0.6 M NaCl 10 mM Tris · HCl (pH 7.5), 1 mM EDTA, 1× Denhardt's reagent, 10% PEG 8000, 50 µg/ml heparin, 0.5 mg/ml yeast tRNA, and 2 ng/µl RNA probe. Probed sections were then covered with silanized glass coverslips. After hybridization, coverslips were removed in warmed 2× SSC. The sections were washed in 2× SSC for 30 min at 50°C and treated with RNase A 20 µg/ml) in 2× SSC at 37°C for 30 min. The slides were then washed for 30 min at 50°C in 50% formamide in 2× SSC followed by 0.07% sodium pyrophosphate in 1× SSC. Slides were developed using a horseradish peroxidase-tyramide signal amplification kit (NEN), visualized with nickel-enhanced 3,3'-diaminobenzidine (DAB), and stained with hematoxylin (Biomeda, Foster City, CA).

Western blot analysis. For analysis of expression of EGPx protein, 100 mg of mouse small intestine (proximal ileum), cecum, and large intestine (proximal colon) were collected and homogenized in 10 mM Tris, 150 mM NaCl, 0.5% Triton X-100, 1% Nonidet P-40, 10 mM NaN3, 1 µg/ml leupeptin, 1 mM para-aminobenzamide, and 0.5 mM PMSF, pH 7.5. Fifty micrograms of intestinal protein were analyzed by 12.5% SDS-PAGE under reducing conditions (100 mM DTT) and electroblotted onto a membrane (Hybond polyvinylidene difluoride membrane, Amersham Life Science). The membranes were blocked with PBS containing 6% (wt/vol) nonfat dry milk at room temperature for 1 h and then incubated for 1 h with either rabbit anti-mouse EGPx antibody or rabbit anti-mouse CGPx IgG at a 1:20,000 dilution. After the membranes were washed in PBS containing 0.05% polyethylene-sorbitan monolaurate, the immunoblots were incubated with the secondary antibody goat anti-rabbit IgG-horseradish peroxidase at a 1:10,000 dilution (Pierce, Rockford, IL) for 1 h at room temperature and developed with Super Signal Western blotting substrate (Pierce). Blots were exposed to X-ray film and quantified by densitometry (PDI). Mouse plasma and kidney were used as controls.

Immunohistochemistry. Human and mouse intestinal tissue were dissected into 27-mm3 sections and placed into 4% paraformaldehyde fixative. Sections were embedded in paraffin, and 5-µm thick sections were prepared on gelatin-coated glass slides. After microwave oven heating of slides for 10 min in 0.01 M citrate buffer, pH 6.0, to abolish endogenous alkaline phosphatase activity, sections were incubated with either rabbit anti-human or -mouse EGPx antibody and rabbit anti-human or -mouse CGPx antibody at 10 µg/ml for 1 h at room temperature. After several washes with PBS, sections were sequentially incubated with biotin-sp-conjugated affinity-purified donkey anti-rabbit IgG at a 1:2,500 dilution (Accurate Chemical and Scientific, Westbury, NY) and streptavidin-alkaline phosphatase conjugate (Calbiochem) at a 1:2,500 dilution for 1 h each at room temperature. Sections were visualized with fast red (Biomeda) and counterstained with hematoxylin. Some sections were visualized with streptavidin-horseradish peroxidase at a 1:100 dilution plus nickel-enhanced DAB. When using horseradish peroxidase, the sections were first treated with 0.3% H2O2 in PBS for 30 min. Rabbit IgG was used as a control (Sigma Chemical, St. Louis, MO).

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Human large intestinal explants synthesize CGPx and EGPx and secrete EGPx. To determine whether the large intestine synthesizes and secretes EGPx, we metabolically labeled normal human large intestinal explants with [35S]methionine for 24 h. Labeled proteins were exhaustively immunoprecipitated from the explants and the medium using our previously described antibodies and analyzed by SDS-PAGE under reducing (100 mM DTT) conditions. These rabbit polyclonal antibodies were raised against purified CGPx and EGPx and are specific and mutually non-cross-reacting (28). Under reducing conditions, immunoprecipitated EGPx and CGPx have apparent molecular masses of 24 and 23 kDa, respectively. As can be seen in Fig. 1A, EGPx and CGPx are present in the explants and EGPx only is present in the medium. The presence of radioactive bands at these specific locations demonstrates that the explants synthesized both EGPx and CGPx, but only EGPx was secreted. Trace amounts of a band immunoprecipitated by anti-CGPx IgG from the medium of the explants are probably due to some cell lysis during culture of these explants (Fig. 1). The ratio of medium to explant for EGPx is much greater than the ratio of medium to explant for CGPx (Fig. 1B). This difference indicates that the amount of EGPx in the medium is due to secretion and not to cell lysis or leakage.


View larger version (19K):
[in this window]
[in a new window]
 


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 1.   A: SDS-PAGE of immunopurified selenoproteins for intestinal explants and medium. Intestinal explants were metabolically labeled for 24 h with [35S]methionine. Extracellular glutathione peroxidase (EGPx) and cellular glutathione peroxidase (CGPx) were immunopurified from explant extracts and culture medium using the indicated rabbit IgG. Reduced proteins were separated by SDS-PAGE, and autoradiofluorographs are presented. B: quantification of EGPx (solid bars) and CGPx (open bars) immunopurified proteins in human large intestinal explants (e) and medium (m) from intestinal explants after 24 h of incubation with [35S]methionine.

EGPx and CGPx mRNA is expressed throughout mouse gastrointestinal tract. To determine the expression pattern of EGPx and CGPx mRNA along the gastrointestinal tract, we isolated total RNA from various mouse intestinal sections and analyzed it using Northern blot analysis. To ensure that equal amounts of mRNA were being analyzed, the same blots were probed with alpha -tubulin and the levels of both EGPx and CGPx were normalized to the level of alpha -tubulin. As can be seen in Fig. 2, the level of EGPx mRNA expression was similar throughout the mouse gastrointestinal tract as measured by Northern blot analysis when normalized to alpha -tubulin. CGPx mRNA expression, when normalized to alpha -tubulin (Fig. 2), appears to increase from the proximal to the distal end of the gastrointestinal tract. Thus EGPx mRNA expression relative to CGPx mRNA expression is higher in the proximal end of the gastrointestinal tract.


View larger version (57K):
[in this window]
[in a new window]
 
Fig. 2.   Quantification of EGPx and CGPx mRNA expression in mouse gastrointestinal tract. Alternating 2-cm sections were taken from the proximal duodenum to the distal colon [the approximate length of gastrointestinal tract measured in adult mice is 35 cm for small intestine and 14 cm for large intestine (Ref. 13)]. Total RNA (10 µg) was resolved by electrophoresis. Through Northern blot analysis, samples were probed with EGPx, CGPx, and alpha -tubulin cDNA probes. Levels of EGPx (open bars) and CGPx (crosshatched bars) normalized to alpha -tubulin are plotted, as is EGPx normalized to CGPx (bullet ).

Localization of EGPx and CGPx mRNA in human and mouse large intestine. Using biotin-labeled anti-sense EGPx and CGPx cRNA probes, we found that only a subset of cells in the large intestine contained EGPx and CGPx transcripts. In human and mouse large intestine, EGPx and CGPx transcripts were localized predominantly to the mature absorptive epithelial cells (Fig. 3). In addition, in mouse small and large intestine, CGPx transcripts were localized to the muscularis (data not shown). Using these conditions and probes, we were unable to demonstrate the presence of either EGPx or CGPx mRNA in human or mouse small intestine (data not shown). To provide additional evidence for the localization of EGPx mRNA, the villus epithelium was gently scraped away from the muscularis in mouse small intestine. EGPx and CGPx mRNA were measured by Northern blot analysis. To ensure that equal amounts of mRNA were being analyzed, the same blot was probed with GAPDH and the levels of both EGPx and CGPx were normalized to the level of GAPDH (Fig. 4). EGPx mRNA was four to five times greater in the samples enriched for the villus epithelium compared with samples enriched for the muscularis (Fig. 4). Using the same blot and procedure, we detected CGPx mRNA to a similar degree in both the villus epithelium and muscularis (Fig. 4). These data support the localization of EGPx mRNA predominantly to the villus epithelium.


View larger version (109K):
[in this window]
[in a new window]
 
Fig. 3.   Expression of EGPx and CGPx mRNA in human and mouse large intestine determined by in situ hybridization. Bright-field photomicrographs of normal human (A, B) and mouse (C, D) large intestine probed with biotinylated EGPx (A, C) and CGPx (B, D) cRNA probes. Intestinal sections were visualized with nickel-enhanced 3,3'-diaminobenzidine (DAB). Brown deposits indicate areas of transcript expression in these hematoxylin-stained sections. Sense controls were completely negative (data not presented). Bar, 100 µm.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 4.   Quantification of EGPx and CGPx mRNA expression in mouse villus epithelium and muscularis. Using a 2-cm section of proximal ileum, we removed villus epithelium from muscularis and analyzed both samples. Total RNA (10 µg) was resolved by electrophoresis. Through Northern blot analysis, samples were probed with EGPx, CGPx, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probes. Levels of EGPx (solid bars) and CGPx (hatched bars) normalized to GAPDH are plotted.

Presence of EGPx and CGPx protein in mouse intestine by Western blot. EGPx and CGPx protein levels in the mouse gastrointestinal tract (proximal ileum segment of small intestine, cecum, and proximal colon segment of large intestine) were determined by Western blot using polyclonal antibodies raised against peptides specific for EGPx or CGPx. As shown in Fig. 5A, EGPx protein was present in all three sampled intestinal sections. Within the gastrointestinal tract, the highest amount of EGPx protein was present in the cecum (Fig. 5B). In comparison, CGPx protein was also present in the three sampled intestinal sections (Fig. 5A).


View larger version (37K):
[in this window]
[in a new window]
 


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 5.   A: expression of EGPx and CGPx protein in mouse gastrointestinal tract. Western blot analysis is shown of proteins present in mouse gastrointestinal tract (proximal ileum segment of small intestine, cecum, and proximal colon segment of large intestine). Samples were probed with rabbit anti-mouse EGPx and CGPx antibody. Mouse plasma and kidney were used as controls. Samples were taken from 1 mouse, and autoradiographs are presented. B: quantification of EGPx and CGPx protein in mouse gastrointestinal tract. EGPx (solid bars) and CGPx (hatched bars) protein levels were determined by densitometry of Western blots with anti-EGPx and anti-CGPx. Protein levels in the various samples [plasma (P), kidney (K), proximal ileum segment of small intestine (S), cecum (C), and proximal colon segment of large intestine (L)] were normalized to plasma for EGPx and to kidney for CGPx.

Localization of EGPx and CGPx protein in human and mouse intestine. EGPx and CGPx protein localization in the mouse and human gastrointestinal tract was determined by immunohistochemistry using affinity-purified antibodies directed against peptides specific for EGPx or CGPx. In human large intestine, EGPx protein is concentrated in mature absorptive epithelial cells and beneath the basolateral membrane of the epithelial layer, consistent with its presence in the extracellular milieu. In contrast, CGPx protein is found only in the mature absorptive epithelial cells and throughout the lamina propria (Fig. 6, A and B). In the mouse large intestine, EGPx and CGPx proteins are predominantly located in the mature absorptive epithelial cells (Fig. 6, C and D). In the mouse large intestine samples, it could not be determined if EGPx protein was present in the space consistent with the extracellular milieu (Fig. 6C). In additional experiments, we were able to demonstrate in mouse small intestine that EGPx protein was present in extracellular compartments, whereas CGPx protein was present only in cellular compartments (Fig. 7, arrows). In addition, CGPx protein was also found in the muscularis of mouse small and large intestines (data not shown).


View larger version (103K):
[in this window]
[in a new window]
 
Fig. 6.   Expression of EGPx and CGPx protein in human and mouse large intestine. Bright-field photomicrographs are shown of normal human and mouse intestine that was fixed in 4% paraformaldehyde and embedded in paraffin. Sections were incubated with rabbit anti-human (A, B) and -mouse (C, D) EGPx IgG (A, C) and CGPx IgG (B, D). Specific antibody binding was visualized by sequential incubations with biotinylated secondary antibody and streptavidin alkaline phosphatase for human sections and streptavidin horseradish peroxidase for mouse sections. Human sections were visualized with fast red, and mouse samples were visualized with nickel-enhanced DAB. Red deposits seen in A and B and brown deposits seen in C and D indicate locations of specific proteins in these hematoxylin-stained sections. Note that red deposits are located below basolateral membrane (A, inset, arrow) suggestive of the presence of EGPx in the extracellular space. In contrast, note the light (clear) region below the basolateral membrane (B, inset, arrow) indicating absence of CGPx in this space in which EGPx is found. Bars, 100 µm.


View larger version (102K):
[in this window]
[in a new window]
 
Fig. 7.   Expression of EGPx and CGPx protein in mouse small intestine. Bright-field photomicrographs of normal mouse small intestine that was fixed in 4% paraformaldehyde and embedded in paraffin. Sections were incubated with rabbit anti-mouse EGPx IgG (A) and CGPx IgG (B). Specific antibody binding was visualized by sequential incubations with biotinylated secondary antibody and streptavidin horseradish peroxidase. Sections were visualized with nickel-enhanced DAB. Brown deposits seen in A and B indicate locations of specific proteins in these hematoxylin-stained sections. These are sections in which epithelial cells are protruding out of the plane of the section. There is a brown halo (A, arrow) around cells indicative of EGPx in the extracellular space. In contrast, there is a light (clear) halo (B, arrow) around cells, indicating the absence of CGPx in the extracellular space. Bars, 100 µm.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

EGPx has been shown to be synthesized and secreted by the human colon adenocarcinoma cell line Caco-2, in addition to kidney and liver cell lines (4). In this study, we have demonstrated that explants of resected normal human large intestine synthesize EGPx and CGPx and secrete EGPx. Northern blots of mouse intestinal mRNA have demonstrated that EGPx transcripts are found throughout the gastrointestinal tract. In situ hybridization studies have shown that EGPx mRNA transcripts are localized to mature absorptive epithelia of human and mouse large intestine. Additional verification for the localization of EGPx mRNA in the villus epithelium in mice was obtained by Northern analysis of samples enriched for villus epithelium. The scraping procedure previously described generally removes the distal villus epithelium, leaving behind the crypts with the muscularis (30). The four- to fivefold greater amount of EGPx mRNA found in the villus epithelium compared with the muscularis may underestimate the relative contribution since the technique may not have completely removed all of the villus epithelium from the muscularis. Western blot analysis of mouse intestinal protein has demonstrated the presence of EGPx protein in small intestine, cecum, and large intestine, with the highest protein levels found in the cecum. With the use of immunohistochemistry, EGPx protein has been found within mature absorptive epithelia in human large intestine and mouse small and large intestine and within the extracellular milieu in human large intestine and mouse small intestine.

Northern analysis of mouse intestinal tissue revealed similar levels of EGPx mRNA expression throughout the entire mouse gastrointestinal tract. Surprisingly, the amount of EGPx protein detected by Western analysis was not similar for the three sections of mouse intestine that were sampled. It is unclear at the present time as to whether this difference is due to changes in rates of synthesis, secretion, or degradation. We were also unable to detect any evidence of EGPx and CGPx mRNA in human and mouse small intestine during in situ hybridization. However, using the same tissue samples, we were able to detect protein by the method of immunohistochemistry. Perhaps the mRNA in these particular samples were unstable under the conditions utilized during in situ hybridization, which prohibited us from detecting the transcripts of both EGPx and CGPx.

Another member of the selenium-dependent GPx family, GPx-GI, has been identified by Chu et al. (11). This cellular selenium-dependent GPx is readily detectable in the rat gastrointestinal tract. Esworthy et al. (15) have reported that GPx-GI and CGPx activities were uniformly distributed in the rat middle and lower gastrointestinal tract and with respect to the villus-to-crypt axis. In addition, GPx-GI activity levels were found to be nearly equal to that of CGPx throughout the rat small intestine and colorectal segments. We were able to detect CGPx mRNA and protein in human and mouse intestinal epithelial cells, in agreement with recent work in rats and mice by Esworthy et al. (15).

The intestinal epithelial cells are composed of rapidly dividing stem cells, which are located in crypts, and differentiated short-lived cells, which are located in villi. It has been demonstrated that the activities of several antioxidant enzymes, including GPx, catalase, superoxide dismutase, and glutathione S-transferase, are present in the intestinal epithelium. However, the exact location of GPx enzyme activity within the mucosal epithelium has variously been reported to range from its predominance in either the distal villus (23), the crypt region (12), or both (15). The antioxidants present in the intestinal epithelium represent the first line of defense against the ingested oxidative toxins and xenobiotics. The villus epithelial cells of the intestine are directly exposed to dietary materials and may therefore be especially susceptible to oxidative damage. Dietary peroxides can be metabolically reduced to hydroxy lipids in the gastrointestinal tract (20). Our data from humans and mice have demonstrated that EGPx and CGPx mRNA are predominantly found in the villus. Their presence in the villus suggests a protective role in this region. CGPx, as an intracellular GPx, would protect the villus epithelial cells, while EGPx would provide extracellular antioxidant protection for the villus. Exogenous glutathione has been shown to protect the intestinal villus from oxidative injury (8, 9, 20, 22). Exogenous glutathione may therefore enhance the effectiveness of these different components of the GPx system (EGPx, CGPx, and GPx-GI) of the gastrointestinal tract.

Inflammatory bowel disease (IBD), such as Crohn's disease and ulcerative colitis, causes chronic injury to the gastrointestinal tract, and studies have shown that antioxidant defenses are altered in these diseases. Studies have demonstrated that the colons of IBD patients produce more oxygen free radicals compared with those of control subjects (17). Free radicals have been implicated in colonic disease, such as ulcerative colitis (25-26), and in vitro studies using enterocytes have shown the damaging effect of exposure to oxidants (10). Considerable debate still exists as to whether these free radicals are involved in the pathogenesis of tissue injury or are generated as a consequence of damaged tissue. The increase in mucosal permeability that results from injury to the epithelium may facilitate the entry of luminal components such as bacteria and bacterial products into the mucosal interstitium, which may ultimately reach the systemic circulation. The clear presence of EGPx in the colon suggests a protective mechanism for reactive oxygen species that have found their way into the extracellular milieu. It has been reported that patients with IBD have an increase in GPx activity in their plasma (17, 29). However, a direct relationship between IBD and EGPx expression in the gastrointestinal tract remains to be determined. Under ordinary circumstances, plasma GPx activity is mainly derived from the kidneys (5). However, ~5-25% of the normal plasma GPx activity appears to be due to nonrenal sources, as seen in the residual activity in the plasma of anephric patients (5, 31). We have presented data that demonstrate secretion of extracellular GPx by intestinal explants. The immunohistochemistry results have demonstrated EGPx protein located outside the epithelial cells, beneath the absorptive epithelial cells in the human large intestine and mouse small intestine, which may suggest a basolateral secretion. The secretion of EGPx by intestinal epithelia may provide extracellular protection within the intestine, in the interstitial space. Intestinal expression of EGPx might be needed as a protective mechanism against lipid peroxides found in this specific area.

This present study has demonstrated the presence of the defense enzyme EGPx in the gastrointestinal tract, and this enzyme along with others may provide protection against oxidant injury. We have demonstrated that selenium-dependent EGPx is synthesized and secreted by human and mouse intestinal epithelia. Its presence in the extracellular milieu of the intestine suggests a localized role in antioxidant defense and/or metabolism of peroxides.

    ACKNOWLEDGEMENTS

We thank Dr. Rodney U. Anderson for providing tissue specimens.

    FOOTNOTES

This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant 2R01-DK-33231.

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. §1734 solely to indicate this fact.

Address for reprint requests: D. M. Tham, Dept. of Pediatrics, Stanford Univ. School of Medicine, 300 Pasteur Drive, Rm. S308, Stanford, CA 94305-5208.

Received 16 June 1998; accepted in final form 31 August 1998.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

1.   Autrup, H., L. A. Barrett, F. E. Jackson, M. L. Jesudason, G. Stoner, P. Phelps, B. F. Trump, and C. C. Harris. Explant culture of human colon. Gastroenterology 74: 1248-1257, 1987[Medline].

2.   Avissar, N., C. Eisenmann, J. G. Breen, S. Horowitz, R. K. Miller, and H. J. Cohen. Human placenta makes extracellular glutathione peroxidase and secretes it into maternal circulation. Am. J. Physiol. 267 (Endocrinol. Metab. 30): E68-E76, 1994[Abstract/Free Full Text].

3.   Avissar, N., J. N. Finkelstein, S. Horowitz, J. C. Willey, E. Coy, M. W. Frampton, R. H. Watkins, P. Khullar, Y. L. Xu, and H. J. Cohen. Extracellular glutathione peroxidase in human lung epithelial lining fluid and in lung cells. Am. J. Physiol. 270 (Lung Cell. Mol. Physiol. 14): L173-L182, 1996[Abstract/Free Full Text].

4.   Avissar, N., E. A. Kerl, S. S. Baker, and H. J. Cohen. Extracellular glutathione peroxidase mRNA and protein in human cell lines. Arch. Biochem. Biophys. 309: 239-246, 1994[Medline].

5.   Avissar, N., D. B. Ornt, Y. Yagil, S. Horowitz, R. H. Watkins, E. A. Kerl, K. Takahashi, I. S. Palmer, and H. J. Cohen. Human kidney proximal tubules are the main source of plasma glutathione peroxidase. Am. J. Physiol. 266 (Cell Physiol. 35): C367-C375, 1994[Abstract/Free Full Text].

6.   Avissar, N., J. R. Slemmon, I. S. Palmer, and H. J. Cohen. Partial sequence of human plasma glutathione peroxidase and immunologic identification of milk glutathione peroxidase as the plasma enzyme. J. Nutr. 121: 1243-1249, 1991[Medline].

7.   Avissar, N., J. C. Whitin, P. Z. Allen, I. S. Palmer, and H. J. Cohen. Antihuman plasma glutathione peroxidase antibodies: immunological investigations to determine plasma glutathione peroxidase protein and selenium content in plasma. Blood 73: 318-323, 1988[Abstract].

8.   Aw, T. Y., and M. W. Williams. Intestinal absorption and lymphatic transport of peroxidized lipids in rats: effect of exogenous GSH. Am. J. Physiol. 263 (Gastrointest. Liver Physiol. 26): G665-G672, 1992[Abstract/Free Full Text].

9.   Aw, T. Y., M. W. Williams, and L. Gray. Absorption and lymphatic transport of peroxidized lipids by rat small intestine in vivo: role of mucosal GSH. Am. J. Physiol. 262 (Gastrointest. Liver Physiol. 25): G99-G106, 1992[Abstract/Free Full Text].

10.   Baker, S. S., and C. L. Campbell. Enterocyte injury by O2-dependent processes (Abstract). Gastroenterology 96: A23, 1989.

11.   Chu, F. F., J. H. Doroshow, and R. S. Esworthy. Expression, characterization and tissue distribution of new cellular selenium-dependent glutathione peroxidase, GSHPx-GI. J. Biol. Chem. 268: 2571-2576, 1993[Abstract/Free Full Text].

12.   Chu, F. F., and R. S. Esworthy. The expression of an intestinal form of glutathione peroxidase (GSHPx-GI) in rat intestinal epithelium. Arch. Biochem. Biophys. 323: 288-294, 1995[Medline].

13.   Cook, M. J. Anatomy. In: The Mouse in Biomedical Research, edited by H. L. Foster, J. D. Small, and J. G. Fox. New York: Academic, 1983, p. 101-120.

14.   Esworthy, R. S., F. F. Chu, P. Geiger, A. W. Girotti, and J. H. Doroshow. Reactivity of plasma glutathione peroxidase with hydroperoxide substrates and glutathione. Arch. Biochem. Biophys. 307: 29-34, 1993[Medline].

15.   Esworthy, R. S., K. M. Swiderek, Y. S. Ho, and F. F. Chu. Selenium-dependent glutathione peroxidase-GI is a major glutathione peroxidase activity in the mucosal epithelium of rodent intestine. Biochim. Biophys. Acta 1381: 213-226, 1998[Medline].

16.   Flohe, L. The selenoprotein glutathione peroxidase. In: Glutathione: Chemical, Biochemical and Medical Aspects, edited by D. Dolphin, O. Avramovic, and R. Poulson. New York: Wiley, 1989, p. 643-732.

17.   Hoffenberg, E. J., J. Deutsch, S. Smith, and R. J. Sokol. Circulating antioxidant concentrations in children with inflammatory bowel disease. Am. J. Clin. Nutr. 65: 1482-1484, 1997[Abstract].

18.   Keshavarzian, A., S. Sedghi, J. Kanofsky, T. List, C. Robinson, C. Ibrahim, and D. Winship. Excessive production of reactive oxygen metabolites by inflamed colon: analysis by chemiluminescence probe. Gastroenterology 103: 177-185, 1992[Medline].

19.   Kingsley, P., J. C. Whitin, H. J. Cohen, and J. Palis. Developmental expression of extracellular glutathione peroxidase suggests antioxidant roles in deciduum, visceral yolk sac, and skin. Mol. Reprod. Dev. 49: 343-355, 1998[Medline].

20.   Kowalski, D. P., R. M. Feeley, and D. P. Jones. Use of exogenous glutathione for metabolism of peroxidized methyl linoleate in rat small intestine. J. Nutr. 120: 1115-1121, 1990[Medline].

21.   Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970[Medline].

22.   Lash, L., T. M. Hagen, and D. P. Jones. Exogenous glutathione protects intestinal epithelial cells from oxidative injury. Proc. Natl. Acad. Sci. USA 83: 4641-4645, 1986[Abstract].

23.   Manohar, M., and K. A. Balasubramanian. Antioxidant enzymes in rat gastrointestinal tract. Indian J. Biochem. Biophys. 23: 274-278, 1986[Medline].

24.   Maser, R. L., B. S. Magenheimer, and J. P. Calvet. Mouse plasma glutathione peroxidase: cDNA sequence analysis and renal proximal tubular expression and secretion. J. Biol. Chem. 269: 27066-27073, 1994[Abstract/Free Full Text].

25.   Parks, D. A., G. B. Bulkely, and D. N. Granger. Role of oxygen-derived free radicals in digestive tract diseases. Surgery 94: 415-422, 1983[Medline].

26.   Rokutan, K., T. Hosokawa, N. Marui, A. Yoshida, K. Nakamura, K. Koyama, A. Aoike, and K. Kawai. Modulation of antioxidant enzyme activities in murine gastric mucosa by aging and nutrition. In: Free Radicals in Digestive Diseases, edited by M. Tsuchiya, K. Kawai, M. Kondo, and T. Yoshikawa. Amsterdam: Elsevier Science, 1988, p. 123-128.

27.   Sprenger, H., L. Konrad, E. Rischkowsky, and D. Gemsa. RNA extraction from gastrointestinal tract and pancreas by a modified Chomczynski and Sacchi method. Biotechniques 19: 340-344, 1995[Medline].

28.   Takahashi, K., N. Avissar, J. C. Whitin, and H. J. Cohen. Purification and characterization of a novel monomeric glutathione peroxidase: a selenoglycoprotein distinct from the known cellular enzyme. Arch. Biochem. Biophys. 256: 677-686, 1987[Medline].

29.   Thomas, A. G., V. Miller, A. Shenkin, G. S. Fell, and F. Taylor. Selenium and glutathione peroxidase status in paediatric health and gastrointestinal disease. J. Pediatr. Gastroenterol. Nutr. 19: 213-219, 1994[Medline].

30.   Van Der Vliet, A., T. J. R. Tuinstra, and A. Bast. Modulation of oxidative stress in the gastrointestinal tract and effect on rat intestinal motility. Biochem. Pharmacol. 38: 2807-2818, 1989[Medline].

31.  Whitin, J. C., D. M. Tham, S. Bhamre, D. B. Ornt, J. D. Scandling, B. M. Tune, O. Salvatierra, N. Avissar, and H. J. Cohen. Plasma glutathione peroxidase and its relationship to renal proximal tubule function. Mol. Gen. Metab. In press.

32.   Yamamoto, Y., Y. Nagata, E. Niki, K. Watanabe, and S. Yoshimura. Plasma glutathione peroxidase reduces phosphatidylcholine hydroperoxide. Biochem. Biophys. Res. Commun. 193: 133-138, 1993[Medline].

33.   Yamamoto, Y., and K. Takahashi. Glutathione peroxidase isolated from plasma reduces phospholipid hydroperoxides. Arch. Biochem. Biophys. 305: 541-545, 1993[Medline].


Am J Physiol Gastroint Liver Physiol 275(6):G1463-G1471
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society