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Research Communication

Immunological Analysis of ß-Thalassemic Mouse Intestinal Proteins Reveals Up-Regulation of Sucrase-Isomaltase in Response to Iron Overload¹

Department of Nutrition, Harvard School of Public Health, Boston, MA 02115

²To whom correspondence should be addressed.

	ABSTRACT

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Maintenance of iron homeostasis must balance the demand for iron due to heme synthesis, which is driven by hematopoiesis, and the restricted intestinal uptake of iron, which otherwise limits absorption of this toxic element. The consequences of perturbed iron homeostasis are witnessed in inherited forms of ß-thalassemia in which erythroid hyperplasia results in enhanced intestinal iron absorption despite tissue iron overload. To gain a better understanding of intestinal factors that are induced when iron homeostasis is disrupted, a panel of monoclonal antibodies that recognize intestinal microvillous membrane proteins of the ß-thalassemic Hbb^d(th3)/Hbb^d(th3) mouse was established. The monoclonal antibodies were screened by differential Western blotting against normal and ß-thalassemic mouse intestine to identify antigens modulated in the disease state. Here we report the initial characterization of one immunoreactive species that is up-regulated in ß-thalassemic mouse intestine and the tentative identification of this antigen as sucrase-isomaltase. Studies in Caco-2 cells revealed the rather unexpected finding that expression of this intestinal hydrolase is increased in response to iron toxicity.

KEY WORDS: • iron • ß-thalassemia • sucrase-isomaltase • Hbb^d(th3) mice

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
ß-Thalassemia is an inherited form of anemia caused by defects in synthesis of the ß-globin chain of hemoglobin. This hemoglobinopathy compromises erythropoiesis, resulting in defective oxygen transport and hemolytic anemia. Erythroid hyperplasia promulgated by this genetic disorder enhances intestinal absorption of iron (Crosby and Conrad 1960 , Fargion et al. 1982 , Pippard et al. 1964 ), a physiologic response that compounds the transfusional iron overload incurred by treatment of this disease (Olivieri and Brittenham 1997 ). A mouse model for this genetic trait was established in 1983 by the discovery of a mutation that resulted in deletion of the murine ß-major globin gene (Skow et al. 1983 ). Like their human counterparts, Hbb^d(th3)/Hbb^d(th3) mice have increased intestinal absorption of iron yet suffer from severe tissue iron overload (Van Wyck et al. 1987 ). Thus, these animals provide an important experimental model for the nontransfusional iron balance disorder manifested by the human disease. To gain a better understanding of intestinal factors induced by the ß-thalassemic condition, a panel of monoclonal antibodies that recognize microvillous membrane proteins of the Hbb^d(th3)/Hbb^d(th3) mouse intestine was established. Here we report the initial characterization of one immunoreactive species that is up-regulated in ß-thalassemic mouse intestine and the tentative identification of this antigen as sucrase-isomaltase. The observation that sucrase-isomaltase activity can be experimentally induced by iron loading Caco-2 cells in culture reveals that expression of this factor is indeed modulated by iron toxicity.

	MATERIALS AND METHODS

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Preparation of microvillous membrane protein and antibody production.

Heterozygous C57BL/6J Hbb^d(th3) mice were obtained from Jackson Laboratories (Bar Harbor, ME) and were given free access to nonpurified diet (RodentChow 5001, Purina Mills, St. Louis, MO). Offspring were scored for homozygosity as described (Whitney 1978 ). For some experiments, intestinal tissue samples from homozygous Hbb^d(th3) animals were generously provided by Dr. Raymond Popp, Oak Ridge National Laboratories, Oak Ridge, TN. Frozen duodenal tissue collected from Hbb^d(th3)/Hbb^d(th3) mice was thawed rapidly at 37°C, and the microvilli were collected by scraping with a razor blade. Microvillous membrane proteins were prepared by the CaCl₂ precipitation method of Schmitz et al. (1973) .

Animal protocols were in compliance with the Guide for the Care and Use of Laboratory Animals and approved by the Harvard Medical Area Standing Committee on Animals. Armenian hamsters (Cytogen, Boston, MA) were immunized intraperitoneally with 50 µg ß-thalassemic mouse microvillous membrane proteins in a 1:1 mixture with RIBI adjuvant (RIBI Immunochem Research, Hamilton, MT) and boosted four times at 3-wk intervals. Twenty-eight days after the last boost, hamsters were injected intraperitoneally with 50 µg ß-thalassemic microvillous membrane protein in saline; the hamsters were killed 3 d later. Splenocytes isolated from the immunized animals were fused with the hypoxantine, aminopterin and thymidine-sensitive murine myeloma cell line P3X63-AG8.653 with polyethylene glycol 1000 at a ratio of 1:1 as described by Harlow and Lane (1988) .

Single cell cloning was performed using conditioned media prepared by culturing splenocytes from 4–5 non-immune hamsters in Dulbecco's modified Eagle's medium (DMEM)³ supplemented with 20% fetal bovine serum (FBS) and 50 mg/L gentimycin for 3 d. The hybridoma cells were diluted in DMEM supplemented with 20% FBS and 50 mg/L gentimycin, with this conditioned medium added at a 1:1 ratio. Single-cell cloning was accomplished by serial dilution such that 200 µL dispensed into 96-well tissue culture plates contained ~1 cell/well. After 21 d, viable hybridoma cells were expanded into 24-well plates with DMEM supplemented with 20% FBS and 50 mg/L gentimycin.

Western blots and screening of hybridomas.

Microvillous membranes were separated on 12% SDS-polyacrylamide gels and transferred to nitrocellulose at 200 mA constant current for 2 h by using a mini-transblot apparatus (BioRad, Richmond, CA). The nitrocellulose was blocked for 30 min with 5% non-fat dry milk in Tris-buffered saline, 0.05% Tween-20 (TBST) and washed once. The blots were then placed on a multiscreen apparatus (BioRad), which partitioned the nitrocellulose into 20 air-tight chambers. Supernatants collected from the hybridomas (600-µL aliquots) were placed in individual chambers for incubation of the blot at room temperature for 1 h. The nitrocellulose was then removed and washed three times with TBST. Alkaline phosphatase-linked rabbit anti-hamster immunoglobulin G (IgG; Jackson Immunoresearch Laboratories, West Grove, PA) was used to detect immunoreactivity at a 1:2000 dilution.

Protein purification and characterization.

Approximately 50% of the total microvillous membrane protein was extracted upon solubilization with 1% Triton X-100, and samples were electrophoresed on a preparative 7.5% SDS-polyacrylamide gel. To visualize bands, the gel was stained with SYPRO orange (BioRad) and the antigenic 117-kDa band was excised. After destaining, the gel fragments were incubated overnight in 0.1% SDS containing 0.4% ß-mercaptoethanol to elute the protein. After microcentrifugation (16,000 x g for 10 min) to remove acrylamide pieces, this extract was concentrated using a Centricon-30 filtration device (Amicon, Beverly, MA); samples were then electrophoresed on a 5% SDS-polyacrylamide gel to further resolve the antigenic peptide band. Western blot analysis using monoclonal antibody 2D3.20 was performed to confirm immunoreactivity; the antigenic protein was judged to be >95% pure on the basis of Commassie staining profiles. Samples were subsequently electrophoresed on a 5% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) membranes; bands were identified by Ponceau S staining and excised. The PVDF-bound protein was subjected to enzymatic digestion with endoproteinase Lys-C as described by Fernandez et al. (1994) . Protein sequence determination of an internal 1364.7-Da proteolytic peptide was performed by the Protein/DNA Technology Center of the Rockefeller University.

Caco-2 cell culture and Northern analysis.

Caco-2 cells (HTB37) were obtained from American Type Culture Collection (Rockville, MD) and cultured in DMEM supplemented with 10% FBS, 50,000 units/L penicillin G, and 50 mg/L streptomycin. To obtain microvillous membrane from iron-loaded cells, the Caco-2 cells were grown in 125 cm² flasks; DMEM-supplemented 65 µmol/L Fe-nitrilotriacetic acid (FeNTA) (1:1 complex) was added for at least 17 d after confluence. Control and iron-loaded cells were lifted by scraping, and brush border membranes were prepared as described by Ekmekcioglu et al. (1996) . For some experiments, Caco-2 cells were grown on 0.4-µm polycarbonate Transwell filters (Costar, Cambridge, MA). The formation of a tight monolayer was monitored by measuring the transepithelial electrical resistance (TEER) by using a Millicell electrical resistance device (Millipore, Bedford, MA). Sucrase activity was measured as described by Dahlqvist (1964) .

Total cellular RNA was extracted from filter-grown Caco-2 cells using RNAzol B (Tel-Test, Friendswood, TX). Samples (60 µg) were electrophoresed on 1% agarose gels with 0.22 mol/L formaldehyde, 20 mmol/L 2-(N-morpholino)propanesulfonic acid, 5 mmol/L sodium acetate and 1 mmol/L EDTA. then transferred to Nytran (Schleicher and Schuell, Keene, NH). After UV-crosslinking, membranes were prehybridized with 50% formamide, 0.75 mol/L NaCl, 0.075 mol/L sodium citrate, 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone and 0.1% Ficoll 400 at 42°C for 3 h. A 420-bp fragment of human sucrase-isomaltase (HSI), generously provided by Drs. Debra Silberg and Peter Traber (University of Pennsylvania, Philadelphia, PA), was radiolabeled with [³²P]dCTP by random priming and used to probe the Northern blot. After overnight incubation at 42°C, the blot was washed twice for 5 min at room temperature with 0.3 mol/L sodium chloride and 0.03 mol/L sodium citrate, then twice for 15 min at 65°C in the same solution with 1% SDS added. The blot was exposed to Kodak XAR film between intensifying screens at -80°C (3 d). The membrane was then stripped by boiling and reprobed with a [³²P]-labeled 700-bp fragment of the human 36B4 ribosomal subunit as a control (overnight exposure).

	RESULTS

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To elucidate factors that are modulated in the ß-thalassemic condition, intestinal microvillous membrane proteins were isolated from Hbb^d(th3)/Hbb^d(th3) mice and used as immunogens to raise a panel of monoclonal antibodies. Hybridoma supernatants were screened by differential Western blotting as shown in Figure 1 . The results of this experiment compare the pattern of immunoreactivity between normal and ß-thalassemic intestinal microvillous membrane proteins, and numerous differences can be noted. Multiple antigens ranging in size from 66 to 250 kD with differential immunoreactivity were observed, and the distinctions between ß-thalassemic and normal intestinal protein expression were too many to be defined specifically. For illustrative purposes, several antigens are highlighted in Figure 1 such as Band I, which is notably prominent in normal mouse intestine (see asterisk, lane 4), but apparently missing from ß-thalassemic microvillous membranes. Bands II and III were detected among ß-thalassemic intestinal proteins (lanes 10–12) but were not found in microvilli from normal mice. Of particular interest is Band IV, which can be detected in both normal and Hbb^d(th3)/Hbb^d(th3) mouse intestinal membranes (see lanes 4, 13, 14 and 17), but which appeared to be more abundant in the latter preparation, suggesting that this factor is up-regulated in the ß-thalassemic condition. Coomassie-staining profiles indicated that this factor was an abundant brush border membrane protein (not shown).

View larger version (112K): [in this window] [in a new window]   Figure 1. Antigens present in normal and ß-thalassemic mouse microvillous membranes recognized by antibodies secreted from various hybridomas. Membrane protein (200 µg) from normal and ß-thalassemic mice was electrophoresed on a 12% SDS-polyacrylamide gel and transferred to nitrocellulose as described in Materials and Methods. The resultant blots were then placed in a multiscreen apparatus to enable multiple hybridomas to be screened at once, each being represented by a lane in the Figure (1–19). Briefly, replicate 600-µL aliquots of supernatants from antibody-producing cells were incubated in individual lanes using the multiscreen apparatus with blots of both control and ß-thalassemic membrane proteins. After 1 h at room temperature, the blots were then washed and incubated with alkaline-phosphatase conjugated anti-hamster immunoglobulin G (IgG) antibody, and immunoreactivity was detected as described in Materials and Methods.

View larger version (106K): [in this window] [in a new window]   Figure 2. Monoclonal antibodies 2C6.1, 2D3.18 and 2D3.20 recognize Band IV, which is more abundant in microvilli of ß-thalassemic mice. Membrane protein (100 µg) from normal and ß-thalassemic mice was electrophoresed on a 7.5% SDS-polyacrylamide gel and transferred as described for Figure 1 . The nitrocellulose blot was incubated with monoclonal antibodies 2C3.1, 2D3.18 and 2D3.20 (diluted 1:20) for 1 h at room temperature.

Table 1

Caco-2 cells were cultured with 65 µmol/L FeNTA to induce iron overload as described by Alvarez-Hernandez et al. (1991) ; under these conditions, cellular iron content is increased more than fourfold. The fact that cellular iron did indeed accumulate under these conditions is demonstrated by the fact that ferritin synthesis was induced as shown in Figure 3 . With the use of an enzymatic assay (Dahlqvist 1964 ), significantly enhanced sucrase activity was consistently observed in microvillous membranes isolated from cells grown in the high Fe medium. Sucrase activity (±SD) was determined to be 23 ± 9.23 and 78.2 ± 8.08 µmol glucose/(mg protein·h) for control and iron-loaded cells, respectively (n = 3; P < 0.0015, t test). The finding that sucrase-isomaltase activity is up-regulated in response to iron toxicity was confirmed by Northern blot experiments demonstrating enhanced expression levels (Fig. 4 ). As previously reported by Alverez-Hernandez et al. (1991) , iron loading Caco-2 cells does not interfere with the overall integrity of the monolayer as monitored by TEER. In agreement with studies by Van Beers et al. (1995) , as Caco-2 cells differentiate and form tight monolayers, HSI expression increases to a maximum level and then declines. However, cells cultured in the presence of 65 µmol/L FeNTA maintained higher HSI transcript levels than control cells (d 28 in Figure 4 inset). Thus, we conclude that the observed increase in Caco-2 cell sucrase activity is due to enhanced levels of HSI mRNA induced by iron toxicity.

View larger version (103K): [in this window] [in a new window]   Figure 3. Ferritin synthesis is up-regulated in iron-loaded Caco-2 cells. Caco-2 cells were grown in media supplements with or without 65 µmol/L Fe-nitrilotriacetic acid (FeNTA) for 17 d to increase cellular iron content as described by Alvarez-Hernandez et al. (1991) . To confirm iron loading, the induction of ferritin synthesis was followed by Western blot analysis as shown. Briefly, cell extracts (150 µg) were electrophoresed on a 10% polyacrylamide gel, transferred to nitrocellulose and probed with polyclonal anti-ferritin antibodies (Boehringer-Mannheim, Indianapolis, IN) at a 1:3000 dilution. Immunoreactivity was detected by enhanced chemiluminescence (ECL) following the manufacturer's directions (Amersham Pharmacia Biotech, Buckinghamshire, UK).

View larger version (23K): [in this window] [in a new window]   Figure 4. Expression of human sucrase-isomaltase (HSI) is induced upon Caco-2 cell differentiation and is greater in iron-loaded cells. Caco-2 cells were grown on 24-mm polycarbonate filters; the transepithelial electrical resistance (TEER) was monitored as described in Materials and Methods. After the TEER achieved a plateau indicative of a fully differentiated monolayer, the cells were treated with (closed circles) or without (open circles) high Fe [media supplemented with 65 µmol/L Fe-nitrilotriacetic acid (FeNTA)]. Total cellular RNA was isolated at the indicated times and the results of Northern blot analysis for HSI and ribosomal subunit 36B4 are shown in the inset.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

An attractive hypothesis to explain this unexpected and intriguing result is that it reflects redox signaling through iron-induced oxidative stress mechanisms (Sen 1998 ). Elevated levels of iron are known to promote the generation of reactive oxygen species, which in turn can activate redox-sensing proteins, typically through key functional cysteine residues. Among these are transcription factors, and it is therefore of particular interest to note that Cdx-2, a homeodomain protein that regulates sucrase-isomaltase gene transcription (Traber 1993 ), dimerizes in a redox-sensitive manner (Suh et al. 1994 ). Sucrase-isomaltase is widely studied because of its differentiation-specific expression in the intestine (Traber and Silberg 1996 ), alterations in its activity promoted by the diabetic condition (Olsen and Korsmo 1977 ) and the glucose-dependent pattern of its regulated expression (Changret et al. 1994 ). The induction of its expression observed in our studies could potentially reflect a general adaptation to nonoxidative metabolism during times of stress, but further experiments are required to determine whether stress inducers other than iron up-regulate sucrase-isomaltase expression. If the up-regulation of sucrase-isomaltase in response to iron toxicity does indeed reflect global changes in gene transcription elicited through redox signaling, then perhaps other antigens recognized by our panel of monoclonal antibodies may be modulated through this mechanism as well. Further characterization of the intestinal factors that are differentially expressed in the ß-thalassemic mouse may also prove of great value in identifying gene products specifically regulated by iron, particularly the regulatory factors responsible for the maintenance of iron balance at the level of intestinal absorption.

	FOOTNOTES

 
¹ Supported by National Institutes of Health grant DK52371 and in part by a Pilot grant from the Clinical Nutrition Research Center at Harvard (NIH grant DK40561). T.A. is a recipient of a fellowship award from the Cooley's Anemia Foundation. M.W.-R. is an Established Investigator of the American Heart Association.

³ Abbreviations used: DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; FeNTA, Fe-nitrilotriacetic acid; HSI; human sucrase-isomaltase; Ig, immunoglobulin; PVDF, polyvinylidene difluoride; TBST, Tris-buffered saline with Tween-20; TEER, transepithelial electrical resistance.

Manuscript received September 11, 1998. Revision accepted January 26, 1999.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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