doi: 10.1152/ajpgi.00198.2004288:G933-G942, 2005. First published 9 December 2004;Am J Physiol Gastrointest Liver Physiol
Bidart, Martin Schlumberger, Alain Virion and Corinne DupuyAgnandji, Renée Ohayon, Jacques Kaniewski, Marie-Sophie Noël-Hudson, Jean-MichelJean-Christophe Sabourin, Virginie Belotte, Stanislas Morand, Sédami Gnidehou, Diane Rabii Ameziane El Hassani, Nesrine Benfares, Bernard Caillou, Monique Talbot,Dual oxidase2 is expressed all along the digestive tract
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Diane Agnandji,1 Renee Ohayon,1 Jacques Kaniewski,1 Marie-Sophie Noel-Hudson,1
Jean-Michel Bidart,2 Martin Schlumberger,2 Alain Virion,1 and Corinne Dupuy1
1Unite 486 INSERM, Universite Paris 11, Faculte de Pharmacie, Chatenay-Malabry
Cedex and 2UMR 8125 CNRS/CEA LRC 29V, Institut Gustave-Roussy, Villejuif, France
Submitted 29 April 2004; accepted in final form 23 November 2004
Ameziane El Hassani, Rabii, Nesrine Benfares, Bernard Cail-lou, Monique Talbot, Jean-Christophe Sabourin, Virginie Belotte,Stanislas Morand, Sedami Gnidehou, Diane Agnandji, ReneeOhayon, Jacques Kaniewski, Marie-Sophie Noel-Hudson, Jean-Michel Bidart, Martin Schlumberger, Alain Virion, and CorinneDupuy. Dual oxidase2 is expressed all along the digestive tract. Am J
Physiol Gastrointest Liver Physiol 288: G933–G942, 2005. Firstpublished December 9, 2004; doi:10.1152/ajpgi.00198.2004.—Thedual oxidase (Duox)2 flavoprotein is strongly expressed in the thyroidgland, where it plays a critical role in the synthesis of thyroidhormones by providing thyroperoxidase with H2O2. DUOX2 mRNAwas recently detected by RT-PCR and in-situ hybridization experi-ments in other tissues, such as rat colon and rat and human epithelialcells from the salivary excretory ducts and rectal glands. We exam-ined Duox2 expression at the protein level throughout the porcinedigestive tract and in human colon. Western blot analysis identifiedDuox2 as the same two molecular species (Mr 165 and 175 kDa) asdetected in the thyroid. It was expressed in all the tissues tested, butthe highest levels were found in the cecum and sigmoidal colon.Immunohistochemical studies showed that Duox2 protein is mainlypresent in these parts of the gut and located at the apical membrane ofthe enterocytes in the brush border, indicating that it is expressed onlyin highly differentiated cells. A Ca2�/NADPH-dependent H2O2-gen-erating system was associated with Duox2 protein expression, whichhad the same biochemical characteristics as the NADPH oxidase inthe thyroid. Indeed, treatment of the thyroid and cecum particulatefractions with phenylarsine oxide resulted in complete calcium de-sensitization of both enzymes. A marked increase in DUOX2 expres-sion was also found during spontaneous differentiation of postconflu-ent Caco-2 cells. The discovery of Duox2 as a novel source of H2O2
in the digestive tract, particularly in the cecum and colon, makes it anew candidate mediator of physiopathological processes.
REACTIVE OXYGEN SPECIES (ROS) have emerged as importantmolecules in many cells and are involved in regulating essen-tial cell functions such as growth and differentiation (2). In thethyroid, H2O2 is the final electron acceptor for the thyroper-oxidase-catalyzed biosynthesis of thyroid hormone at the api-cal surface of the thyrocytes (28).
A functional NADPH oxidase, generating H2O2 in a Ca2�-dependent manner, has been solubilized from pig thyroid (13),and a flavoprotein with an apparent molecular mass of �180kDa has been purified from it (9). Microsequences were used toclone its porcine and human partial cDNAs (9). The full-length
cDNA, encoding a 1,548-amino acid protein known as thyroidoxidase 2 (Thox2), has subsequently been cloned from humanthyroid (5). Its sequence is 83% similar to that of the Thox1,which is also expressed in the thyroid gland (5). Thox1 and 2proteins and THOX1 and 2 genes are also known as dualoxidases1 and 2 (Duox1 and 2) and DUOX1 and 2 genes.
Duox1 and Duox2 are the long homologs of gp91phox, thecatalytic core of phagocytic NADPH oxidase that generatessuperoxide when phagocytic cells ingest bacteria. They belongto a new family of seven NOX/DUOX genes, which encodeseven different NADPH oxidases and have differing mRNAtissue expressions (19). Although a functional Duox-basedH2O2-generating system has not yet been reconstituted (6), theessential role of Duox2 in thyroid hormone synthesis has beenconfirmed by the recent observation of permanent and severecongenital hypothyroidism in a patient with a biallelic inacti-vating mutation in the DUOX2 gene (24).
DUOX2 gene expression is not restricted to the thyroid, andit has also been found in the rat colon by RT-PCR (10).Recently, Leto et al. (11) reported high DUOX2 mRNA ex-pression in the salivary gland and rectum by Northern blotanalyses and visualized DUOX2 expression in rectal epithelialcells by in-situ hybridization experiments. In the same study,small amounts of DUOX2 mRNA were detected by Northernblot analysis in the cecum and ascending colon. We presenthere data demonstrating that the Duox2 protein is expressed inall segments of the porcine digestive tract, but to a particularlymarked degree in the large intestine. This expression wasassociated with NADPH-dependent H2O2-generating activitythat has the same biochemical characteristics as thyroidalNADPH oxidase. We also show that the Duox2 protein isexpressed in the human colon, small intestine, and duodenumas well as in a human colon adenocarcinoma cell line (Caco-2).
MATERIALS AND METHODS
Cell culture. Cultured human colonic adenocarcinoma Caco-2cells, kindly donated by Dr I. Beau (INSERM U 510, Chatenay-Malabry, France), were grown in DMEM (high glucose) medium(Invitrogen) supplemented with 15% FCS, 1% penicillin, 1% strep-tomycin, and 1% Fungizone. The cells were maintained at 37°C in a10% CO2-90% air mixture. The culture medium was changed daily.
Preparation of the particulate fractions. Fresh tissues from twopigs were obtained from Institut National de la RechercheAgronomique and conveyed to the laboratory on ice. Normal humancolon tissues were obtained from surgical specimens at Institut
* R. Ameziane El Hassani and N. Benfares contributed equally to this study.Address for reprint requests and other correspondence: C. Dupuy, INSERM
Unite 486, Faculte de Pharmacie, 92296 Chatenay-Malabry Cedex, France(E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Am J Physiol Gastrointest Liver Physiol 288: G933–G942, 2005.First published December 9, 2004; doi:10.1152/ajpgi.00198.2004.
Gustave Roussy in accordance with local and national ethical lawsand conveyed to the laboratory on ice. All procedures were carried outat �4°C. The porcine digestive tract was divided into eight segments:stomach, duodenum, jejunum, ileum, cecum, sigmoidal colon, floatingcolon, and rectum (Fig. 1). The tissues were cut into small pieces andsuspended in three volumes of 0.25 M sucrose, 50 mM phosphatebuffer, pH 7.20, 1 mM EGTA, 2 mM MgCl2, and protease inhibitors(5 �g/ml leupeptin, 0.15 mg/ml benzamidine, 5 �g/ml aprotinin, 1�g/ml pepstatin, and 16 mg/ml PMSF). The suspension was thenhomogenized with an Ultra-turrax for 2 min. The homogenate wasfiltered through six layers of cheesecloth and centrifuged at 500 g for15 min in a Sorvall SS34 rotor. The supernatant obtained was thencentrifuged at 3,000 g for 15 min in the same rotor. The pellet waswashed twice with three volumes of buffer A (0.25 M sucrose, 50 mMphosphate buffer, pH 7.20, 2 mM MgCl2, and protease inhibitors) andcentrifuged at 3,000 g for 15 min. The final pellet was gentlysuspended in 0.5 volumes of buffer A using a glass/Teflon potterhomogenizer to provide the particulate fraction.
RNA isolation and Northern blot analysis. RNA was extracted fromall the tissues and from the Caco-2 cells by the method of Chomczyn-ski and Sacchi (4). Northern blot analyses were performed as previ-ously described (9). Final washes were carried out at 60°C in 0.1 �SSC, 0.1% SDS (1 � SSC � 0.15 M NaCl, 15 mM sodium citrate).The porcine cDNA probe used was DUOX2 cDNA prepared byRT-PCR, using total RNA from pig thyroids. The sense and antisenseDUOX2 primers, designed on the basis of the 3�-untranslated region(UTR) of the DUOX2 cDNA, were 5�-CACTTCAGGCCTTAGCT-GGA-3� and 5�-GACCAAACGAATCTAGAGCA-3�, respectively.The human cDNA probe used was DUOX2 cDNA prepared byRT-PCR using total RNA from human thyroids. The sense andantisense DUOX2 primers, designed on the basis of the 3�-UTR of theDUOX2 cDNA, were 5�-TGGCAGGCGTGGCAAGCAAA-3� and5�-CACATCAGTGGTTGCTTCTA-3�, respectively. The cDNAprobes were �-32P-labeled by random priming extension using a kit(Amersham). Membranes were analyzed by electronic autoradiogra-phy using INSTANTIMAGER (Packard).
RT-PCR experiment. Total RNA (2 �g) was treated with 15 UThermoScript RT (Invitrogen) in 20 �l PCR buffer for 90 min at 60°Caccording to the manufacturer’s protocol. The control was run withoutthe RT. The porcine DUOX2 cDNA and DUOX1 cDNA were ampli-fied by 30 temperature cycles (95°C, 5 s; 62°C, 10 s; 72°C, 2 min) ina GeneAmp 2400 temperature cycler (Perkin Elmer) in 50 �l pre-
warmed PCR buffer containing 1 �l of Advantage 2 polymerase Mix(Clontech), 250 nM of each sense and antisense oligonucleotideprimer, and 200 �M of each dNTP. Glyceraldehyde 3-phosphatedehydrogenase (G3PDH) cDNA was amplified in parallel. The senseand antisense PCR primers for porcine DUOX2 were designed fromthe porcine DUOX2 cDNA sequence and were 5�-CACTTCAGGC-CTTAGCTGGA-3� and 5�-GACCAAACGAATCTAGAGCA-3�, re-spectively. The amplified product was expected to contain 1051 bp.The sense and antisense PCR primers for porcine DUOX1 cDNA were5�-TACAAGTCAGGACAGTGGGTG-3� and 5�-CGCAGTGCCT-CGCATTTGTC-3�, respectively, and the amplified product was ex-pected to contain 954 bp. The sense and antisense primers for G3PDHwere 5�-ACCACAGTCCATGCCATCAC-3� and 5�-TCCACCAC-CCTGTTGCTGTA-3�, respectively, and the amplified product wasexpected to contain 452 bp.
Cloning of the human extracellular domain of Duox2 and bacterial
expression. The extracellular domain encompassing the G21-L589
fragment of the human Duox2 (ECD) was produced in Escherichia
coli by pTrcHis TOPO TA expression kit (Invitrogen). The expressionof the protein was induced by adding 1 mM isopropyl-�-D-thiogalac-topyranoside to the bacterial culture for 4 h. After being centrifugedat 4,000 g for 20 min, the bacterial pellet was resuspended in 20 ml ofthe lysis buffer (50 mM HEPES-NaOH, pH 7.5, 0.5 mM NaCl, 1 mMPMSF, and 5 mM DTT) containing 0.35 mg/ml lysozyme and soni-cated for 10 min on ice. One milligram of DNAse I was added, and thesuspension was shaken for 1 h at 37°C. The bacterially expressedprotein located in inclusion bodies was obtained by centrifuging at30,000 g for 30 min at 4°C. The pellet was washed twice with PBScontaining 1% Triton X-100 and resuspended in 3 ml 50 mM HEPES-NaOH, pH 7.5, 6 M guanidine/HCl, 1 mM DTT, and 0.2% sarkosyl.Proteins were loaded onto a nickel chelater column (Probond, Invitro-gen) preequilibrated in buffer P {20 mM sodium phosphate buffer, pH7.8, containing 4 M urea, 0.2% sarkosyl, 2 mM 3-[(3-cholamidopro-pyl)dimethylammonio]-1-propanesulfonate (CHAPS), and 0.5 MNaCl}. The column was washed stepwise using buffer P at pH valuesof 6, 5.3, and 4. The bound, His-tagged protein was eluted with 50mM EDTA, pH 7.6, and concentrated 10-fold on centricon 50 (Ami-con) before loading onto a 4–12% polyacrylamide gel. After electro-phoresis, the SDS-PAGE gel was transferred into a 0.3 M solution ofCuCl2 and shaken for 2 min. The band of interest, visualized on ablack background, was cut out, placed in a syringe, and forced throughthe nozzle without the needle into a second syringe according to themethod previously described (26). This was repeated five times. Thegel material was collected in a 2-ml Eppendorf tube with 20 mMsodium phosphate buffer, pH 8, containing 4 M urea and 100 mMEDTA. The mixture was vortexed and incubated at room temperaturefor 15 min and then poured onto a small column (Pierce). The proteinwas eluted from the gel by adding 20 mM sodium phosphate buffer,pH 8, containing 0.4 M urea and 0.01% sarkosyl and concentrated100-fold on centricon 50 (Amicon).
Western blot analysis. Protein samples (40 �g) were suspended inthe sample buffer (2% SDS, 5% �-mercaptoethanol, and 10% glyc-erol), and SDS-PAGE and immunoblot analyses were performed aspreviously described (3). Briefly, an anti-Duox antibody raised againsta 14-amino acid peptide encompassing the L410-T423 portion ofhuman Duox2, which is 100% conserved in porcine Duox2, was usedto probe the immunoblots. A previous study (23) investigating thespecificity of this antipeptide toward porcine Duox1 and Duox2proteins by Western blot analysis has shown that it essentially recog-nizes the pig Duox2 protein. A rabbit polyclonal antibody was raisedagainst the Glu639-Arg1039 fragment of Duox2 produced in E. coli bypTrcHis TOPO TA expression kit (Invitrogen). It was produced byEurogentec (Seraing, Belgium). This antibody was used to probe theimmunoblot of human proteins at a dilution of 1:5,000. It was used ata dilution of 1:200 in immunohistochemistry experiments on humantissues. Monoclonal antibodies raised against the human ECD wereprepared as described in the following protocol. BALB/c mice wereFig. 1. Segmentation of the porcine gut.
G934 DUAL OXIDASE2 EXPRESSED IN THE DIGESTIVE TRACT
injected subcutaneously with 18 �g ECD in 100 �l complete Freundadjuvant, followed by three booster injections with 18, 20, and 50 �gECD in 100 �l incomplete Freund’s adjuvant (Difco) at 3-, 4-, and1-mo intervals, respectively. Finally, mice were injected intraperito-neally with 50 �g ECD in 100 �l incomplete Freund’s adjuvant. Tendays later, their spleens were harvested and hybridoma was prepared.The hybridoma supernatants were evaluated by Western blot analysison membrane extract of the stable human DUOX2-expressing HEK-293 cell line established using the Flp-in system (Invitrogen). Isotyp-ing test revealed that MAbs were IgG2b isotypes. The monoclonalantibody was used to probe the immunoblot at a dilution of 1:500.
Immunohistochemistry. Immunohistochemistry was performed onDuboscq-Brasil-fixed, paraffin-embedded tissue blocks of tissue sam-ples as previously described (3). Normal human tissues were fixed inacetic acid, formaldehyde, and alcohol. Negative controls were pre-pared by incubating tissues with preimmune antisera.
Construction of the human ECD of Duox2 deletions. Deletions ofvarious regions of the ECD were created using the “QuickChange”site-directed mutagenesis kit from Stratagene, with the ECD-pTrcHis-TOPO vector as a template. The �21–122 deletion was created using5�-GATGACGATAAGGATCCAACCCTTGGTTGCCCCGCCGAG-TTCCTCAAC-3� and 5�-GTTGAGGAACTCGGCGGGGCAACCA-AGGGTTGGATCCTTATCGTCATC-3� as mutagenic oligonucleo-tides. The oligonucleotides used for the �123–224 deletion were5�-GACGTGGTGAGCGTGGAAACGCCCCCCGACCCCGCCAC-CGGGCAGAAC-3� and 5�-GTTCTGCCCGGTGGCGGGGTCG-GGGGGCGTTTCCACGCTCACCACGTC-3� The oligonucleotidesused for the �225–326 deletion were 5�-AACCCCCTGCTCATGT-GGGCGGCGGAATTTGTGGTGGCCTCTGAGCAG-3� and 5�-CT-GCTCAGAGGCCACCACAAATTCCGCCGCCCACATGAGCAG-GGGGTT-3�. The oligonucleotides used for the �327–424 deletion were5�-TTCCTAGACCCCAGCATCTCCCCGTATGTGGCCAGCAG-CATCCAACGT-3� and 5�-ACGTTGGATGCTGCTGGCCACAT-ACGGGGAGATGCTGGGGTCTAGGAA-3�. All deletions were con-firmed by sequencing.
Stable cell transfection. The stable human DUOX1- and DUOX2-expressing cell lines were established using the Flp-In system (In-vitrogen). The protocol accompanying the kit was used withoutmodification. Human DUOX1 and DUOX2 cDNAs were subcloned inpcDNA5/FRT vector (Invitrogen) designed for use with the Flp-InSystem. When cotransfected with the pOG44 Flp recombinase ex-pression plasmid into a Flp-In-293 cell line, the pcDNA5/FRT vectorcontaining the DUOX1 or DUOX2 cDNA was integrated in a Flprecombinase-dependent manner into the genome. Stable cell lineswere established by exposure to 100 �g/ml hygromycin.
Preparation of the plasma membrane-enriched fraction from pigthyroid gland. The preparation of plasma membranes was prepared asdescribed previously (21).
Determination of protein content. Protein concentration was deter-mined by the Bradford method (1).
Measurement of NADPH oxidase activity. Particles were incubatedat 30°C in 1 ml 200 mM sodium phosphate buffer, pH 7.4, containing1 mM sodium azide, 0.5 mM CaCl2, 0.4 mM EGTA, 0.1 �M FAD,and 0.1 mM NADPH. Eight aliquots (100 �l) of each sample werecollected at time intervals between 0 and 20 min and mixed with 10�l 3 N HCl to stop the reaction and destroy the remaining NADPH.Fluorescence (excitation: 360 nm, emission: 460 nm) was measured ina Perkin Elmer MPF 43A spectrofluorimeter after adding to eachaliquot 2 ml 200 mM sodium phosphate buffer, pH 7.8, containing0.25 �M scopoletin and 5 �g/ml horseradish peroxidase. The con-centration of H2O2 was directly correlated to the concentration ofoxidized (nonfluorescent) scopoletin.
Treatment of the particulate fraction with phenylarsine oxide. Theparticulate fractions were treated as previously described (14). Briefly,the particulate fraction (1 ml, 100–120 �g/ml) was preincubated for10 min at 0°C in 50 mM sodium phosphate, pH 7.25, containing 0.5mM CaCl2, 0.4 mM EGTA, 1 mM NaN3, and 10 �M FAD and 10 �l
freshly prepared phenylarsine oxide (PAO) solution (at various con-centrations in DMSO). The preincubations were stopped by centri-fuging. The particulate fraction was pelleted by centrifuging at 9,000g at 4°C for 10 min in a Hettich 30 RF centrifuge. The pellet waswashed once with 2 ml sodium phosphate buffer, pH 7.25, containing0.5 mM CaCl2, 0.4 mM EGTA, 1 mM NaN3, 10 �M FAD, and 1 mMCHAPS. The final pellet was resuspended in 50 �l of 200 mM sodiumphosphate buffer, pH 7.25, containing 0.5 mM CaCl2, 0.4 mM EGTA,1 mM NaN3, and 10 mM CHAPS, and the residual activity wasmeasured immediately. The final pellet was resuspended as before butwithout Ca2� and with 7.5 mM EGTA to study the effect of theabsence of Ca2�.
Solubilization. Particulate fractions of porcine sigmoidal colonwere thawed on ice and added with 1% Triton X-100 or 2 M KCl orboth, respectively. The suspensions were gently stirred for 60 min at4°C and centrifuged at 200,000 g for 30 min. The supernatant wasremoved, frozen quickly in liquid nitrogen, and stored at 80°C untilused. The pellet was suspended in the same volume of 50 mM sodiumphosphate, pH 7.2, containing 2 mM MgCl2 and 0.25 M sucrose plusprotease inhibitors (5 �g/ml leupeptin, 0.15 mg/ml benzamidine, 5�g/ml aprotinin, 1 �g/ml pepstatin, and 16 mg/ml PMSF), frozenquick in liquid nitrogen, and stored at 80°C until used.
RESULTS
Detection of porcine DUOX2 along the digestive tract byWestern blot analysis. Immunoblot analysis of membrane pro-teins from different tissues of the digestive tract shows that theprotein Duox2 was expressed in all the tissues tested, but to amuch greater extent in the stomach, cecum, and sigmoidalcolon (Fig. 2).
Western blot analysis showed that membrane proteins dis-played Duox2 as two bands at 165 and 175 kDa, respectively,as previously reported for the pig thyroid (23). These twoproteins correspond to two differently N-glycosylated forms ofDuox2 (6). It was shown that only the more highly glyco-sylated form was resistant to endoglycosidase H digestion,indicating its passage through the Golgi apparatus and alsosuggesting that it constitutes the mature form involved in theactive NADPH oxidase at the plasma membrane (6). Asobserved in the thyroid, the 165-kDa form, which is theprecursor of the 175-kDa form, was more abundant. Preincu-bating the antibody with an excess of synthetic peptide pre-vented the labeling of these proteins (data not shown).
Expression of Duox2 protein in digestive tissues. Figure 3shows an immunohistochemical comparison of Duox2 proteinexpression in porcine thyroid and cecum. As previously shownin human thyroid (3), Duox2 protein was detected at the apicalmembrane of thyrocytes (Fig. 3A). Unlike human thyroid, theporcine tissue was homogenously stained and positive cellswere contiguous, indicating that the mature form of Duox2 was
Fig. 2. Western blot analysis of dual oxidase (Duox)2 from particulate frac-tions of gastrointestinal tissues. Particulate proteins (40 �g) from stomach (1),duodenum (2), ileum (3), jejunum (4), cecum (5), sigmoidal colon (6), floatingcolon (7), and rectum (8) were processed as described in MATERIALS AND
METHODS. Immunoblot analysis was performed with the antipeptide raisedagainst the 14-amino acid peptide encompassing the L410-T423 portion ofporcine Duox2. Particulate proteins (40 �g) from thyroid (lane 9) were used asthe control.
G935DUAL OXIDASE2 EXPRESSED IN THE DIGESTIVE TRACT
expressed to a greater extent in porcine tissue. This finding wasin accordance with previous findings showing that the 175-kDaform is specifically detected in a porcine thyroid fractioncontaining 10–20 times more NADPH oxidase activity thanthe human particulate fraction (21). In the porcine digestivetract, immunohistochemistry revealed intense staining at theapical surface of the epithelial cells of the cecum, with greaterexpression at the surface epithelium (Fig. 3, B and C). Nostaining was seen at the bottom of the crypt. These findingsindicated that Duox2 protein was only expressed in highlydifferentiated cells.
The comparison of immunostaining for Duox2 on serialtissue sections from different gastrointestinal tissues is shownin Fig. 4. In these tissues, staining was observed at the brush
border of the enterocytes, confirming that expression of Duox2was restricted to highly differentiated enterocytes. At highmagnification, immunostaining was also detected in the perinu-clear zone, probably corresponding to the presence of Duox2 inthe Golgi apparatus during maturation (Fig. 4B).
Analysis of DUOX2 mRNA expression along the digestivetract. We studied DUOX2 mRNA expression in the sametissues by Northern blot analysis and compared its expressionto that of the protein. Northern blot analysis detected higherlevels of DUOX2 mRNA in the porcine cecum than in thethyroid. The DUOX2 transcript was also abundant in thesigmoidal colon (Fig. 5A). These results matched the protein-expression profile. On the other hand, DUOX2 mRNA was notdetected by Northern blot analysis in the other tissues, eventhose in which the Duox2 protein appeared to be well ex-pressed, such as in the stomach and floating colon. DUOX2mRNA was, however, detectable by RT-PCR (Fig. 5B), and itwas concluded that DUOX2 transcript levels must vary con-siderably in different tissues and not necessarily be related toprotein expression. We also compared DUOX1 and DUOX2mRNA expression in gastrointestinal and thyroid tissues. Forthis purpose, PCR experiments were performed to generateamplification products of similar size with oligonucleotides inthe specific 3�-UTR of DUOX1 and DUOX2 mRNAs. Figure5B shows that DUOX1 mRNA was not at all, or only veryweakly, expressed in gastrointestinal tissues, whereas it wasclearly detected in the thyroid gland.
Expression of Duox2 protein in human tissues. Figure 6Ashows an immunohistochemical comparison of Duox2 proteinexpression in human colon, small intestine, and duodenum.Immunohistochemistry showed that staining was more pro-nounced on the surface of epithelium. As observed with theporcine colon, the labeling was much more pronounced on theapical membrane. Western blot analysis of surgically removedspecimens from four patients showed that membrane proteinsdisplayed Duox2 as a band at 165 kDa (Fig. 6B). A 165-kDaDuox1/2 protein had already been observed in the 100,000 gpellet from normal human thyroid and after transient transfec-tion of nonthyroid cells (3). The 175-kDa form was notdetectable in human colon tissue, as had previously beenreported in the human thyroid tissue (3).
Measurement of NADPH-dependent formation of H2O2 inporcine gastrointestinal tissues. The expression of a 175-kDaform of Duox2 suggested the presence of a functional Thoxin the gastrointestinal porcine tissues. To confirm this hy-pothesis, we measured the ability of the particulate fractionfrom these tissues to generate H2O2 in the presence ofNADPH. Thyroid NADPH oxidase requires micromolarconcentrations of calcium to acquire a functional conforma-tion and to generate H2O2 (7, 21, 25). Duox2 contains twoCa2�-binding motifs, which could be involved in the directactivation of the H2O2 generator by calcium. Consequently,the NADPH-dependent H2O2-forming activities of gastro-intestinal particulate fractions were measured in the pres-ence and in the absence of calcium. As shown in Fig. 7,particles from all the tissues incubated with NADPH gen-erated H2O2; this was Ca2� dependent as in the thyroidparticulate fraction (7). The H2O2-generating activity wascorrelated to Duox2-protein expression and was muchhigher in cecum and helicoidal colon tissues, where thehighest levels of Duox2 were also found by Western blot
Fig. 3. Expression and localization of Duox2 proteins in porcine thyroid (A)and cecum (B, C). A: immunostaining is localized in the apical membrane offollicular cells (magnification �100). B and C: immunostaining for Duox islocalized at the surface of the cecum epithelium (arrows). No staining isobserved at the bottom of the crypt (magnification �50).
G936 DUAL OXIDASE2 EXPRESSED IN THE DIGESTIVE TRACT
analysis (Fig. 2). The H2O2-generating activity observed instomach was only partly dependent on calcium. This couldbe due to the partial proteolysis of the H2O2-generatingsystem during the preparation of the particulate fractionfrom this tissue, which contains particularly high levels ofproteases. Indeed, we had previously observed that theNADPH oxidase from the thyroid is fully desensitized toCa2� after limited proteolysis by �-chymotrypsin (8).
Solubilization of the NADPH oxidase. To determine thesubcellular distribution of the H2O2-generating system, theporcine colon particulate fractions were treated with 2 M KClwith the nonionic detergent Triton X-100 or with both. Figure8A shows that NADPH-dependent, H2O2-generating activitywas solubilized by a high concentration of salt in the presenceof detergent, as previously demonstrated for pig thyroid plasmamembrane (13). The enzyme was not extracted from theparticulate fractions by either Triton X-100 or KCl alone. Aspreviously observed, the enzymatic activity could only bemeasured after overnight dialysis (13). In a previous study, weshowed that it took at least 6 h to restore the NADPH-dependent, H2O2-generating activity, suggesting that either aconformational change of the protein or a reassociation be-
tween different components extracted by the solubilizationprocedure had occurred (13). Western blot analysis showedthat the Duox2 protein was solubilized under the same condi-tions (Fig. 8B), indicating that the NADPH-dependent, H2O2-generating activity measured under these conditions could beascribed to the Duox2.
Effect of PAO on H2O2 formation. Trivalent arsenical PAOreacts to form stable dithioarsine rings with two thiol groupsthat are either adjacent to each other in the protein sequenceor close together in the folded protein (i.e., vicinal dithiols).In a previous study of thyroidal NADPH oxidase (14), it wasshown that PAO simultaneously caused partial inactivationof the Ca2�-stimulated enzyme and partial activation of thebasal activity, resulting in the complete desensitization ofthe enzyme activity to Ca2�. These findings suggest thatthiol groups are involved in the control of thyroid NADPHoxidase by Ca2� and also provide evidence that thyroidalNADPH oxidase differs from cytochrome b-558, the well-characterized neutrophilic NADPH oxidase, which re-sponded differently to PAO treatment (20). To confirm thatthe NADPH/Ca2�-dependent H2O2 generator identified inall segments of the digestive tract was the same as thyroidal
Fig. 4. Immunostaining for Duox in porcinegastrointestinal tissues. A: immunostaining forDuox in duodenum tissue. Heavy Duox immu-nostaining staining is found in the plasma cellmembrane. Staining is also observed in thecytoplasm (magnification �50). B: immuno-staining for Duox in cecum tissue. Heavy Duoximmunostaining is found both in the cytoplasmand in the plasma cell membrane of the surfaceepithelium. Note the perinuclear location of theimmunostaining (white arrows) (magnification�400). C: immunostaining for Duox in sigmoi-dal colon tissue. Heavy Duox immunostainingis found both in the cytoplasm and in the brushborder of the epithelial cells of the colon (whitearrow; magnification �400). D: immunostain-ing for Duox in floating colon tissue. Heavystaining is observed at the surface of the epi-thelium, both in the cytoplasm and in the cellbrush border (magnification �200). E: immu-nostaining for Duox in rectum tissue. Heavyimmunostaining is found in the cytoplasm andplasma cell membrane (magnification �200).
G937DUAL OXIDASE2 EXPRESSED IN THE DIGESTIVE TRACT
NADPH oxidase, we studied the effects of PAO on theH2O2-generating activity of the particulate fraction of ce-cum (Fig. 9A). The initial rate of H2O2 formation catalyzedby PAO-treated membranes, measured in the absence ofCa2�, increased with the concentration of PAO until aplateau was reached at 12 �M PAO, whereas a symmetricalcurve was obtained for the PAO-induced partial inhibitionof the Ca2�-activated enzyme. Consequently, at PAO con-centrations higher than 10 –12 �M, the partially inhibitedNADPH oxidase became fully Ca2� independent (Fig. 9B).Once again, this desensitization to Ca2� of the PAO-treatedNADPH oxidase was the same as that observed with thy-roidal NADPH oxidase (14).
Expression of Duox2 in a human colon adenocarcinoma cellline (Caco-2). Caco-2 cells spontaneously undergo differenti-ation in postconfluent cultures. Due to their differentiationpotential, these cells constitute a widely used model of entero-cytic differentiation and function. The DUOX2 mRNA expres-sion of postconfluent Caco-2 was analyzed, and marked induc-tion was seen at day 7 (Fig. 10A). The expression of DUOX2mRNA was not detected in exponentially growing nonconflu-ent cultures. To investigate Duox2 protein expression in
Caco-2 cells, we used monoclonal antibodies prepared againstthe human extracellular domain of Duox2. The deletion anal-ysis showed that the monoclonal antibodies recognized theregion between amino acids 123 and 224 (Fig. 10B). Cloningof human DUOX1 and DUOX2 full-length cDNAs permittedus to stably express each protein in HEK293 cells and toevaluate the specificity of this antibody toward human Duoxproteins using Western blot analysis (Fig. 10C). The monoclo-nal antibody, which did not detect porcine Duox2 (lane 3),cross-reacted with both human Duox proteins (lanes 1 and 2)but essentially recognized the human Duox2 (lane 2). Immu-noblot analysis of membrane proteins from Caco-2 cellsshowed that the Duox2 protein was detected at day 10 (Fig.10D). This expression was correlated with an increase in aCa2�-dependent, H2O2-generating activity measured at day 10(Fig. 10E).
Fig. 6. Duox2 expression in human colon. A: 1, immunostaining for Duox inhuman colon. Heavy Duox immunostaining is found both in the cytoplasm andin the brush border of the epithelial cells of the colon (magnification �200). 2,Immunostaining for Duox in small intestine (magnification �20). 3, Immuno-staining for Duox in duodenum (magnification �20). B: Western blot analysisof Duox2 from particulate fractions of human colon. Particulate proteins (40�g) from operative pieces of 4 different patients (lanes 1–4) were processed asdescribed in MATERIALS AND METHODS. Immunoblot analysis was performed asdescribed in MATERIALS AND METHODS with the antibody raised against theGlu639-Arg1039 fragment of Duox2.
Fig. 5. Expression profile of DUOX2 mRNA in gastrointestinal tissuesdetected by Northern blot analysis (A) and RT-PCR (B). Tissue distributionof DUOX1 mRNA determined by RT-PCR (B). A: 20 �g total RNA fromileum (lane 1), sigmoidal colon (lane 2), cecum (lane 3), floating colon(lane 4), duodenum (lane 5), rectum (lane 6), stomach (lane 7), and thyroid(lane 8) were analyzed by Northern blot analysis with the 3�-untranslatedregion probe, as described in MATERIALS AND METHODS. Bottom shows the28S and 18S ribosomal RNA bands detected by methylene blue staining. B:ethidium bromide-stained 1% agarose gel of PCR-generated DNA tem-plates. PCR was performed using single-stranded cDNA generated byreverse transcription of RNAs from the same tissues as template, asdescribed in MATERIALS AND METHODS. G3PDH was amplified to check theintegrity of the cDNA.
G938 DUAL OXIDASE2 EXPRESSED IN THE DIGESTIVE TRACT
DUOX genes were identified in the thyroid gland and werefound to be essentially expressed in this tissue (5, 9). However,expression of the DUOX2 gene is not restricted to the thyroidcell, and DUOX2 mRNA was also found in the rat colon byRT-PCR (10). Recently, Geiszt et al. (11) have published dataconcerning the expression of the DUOX2 transcript in thesalivary glands and rectum detected by Northern blot analysisand in situ hybridization experiments. They found only verylow levels of DUOX2 mRNA in the other gastrointestinaltissues. The results of the present study provide new dataconcerning the expression of the DUOX2 gene in the digestivetract in terms of protein levels. By using an antibody directedagainst the NH2-terminal part of the protein, we showed byWestern blot experiments that Duox2 is not only expressed inthe rectum, but also throughout the digestive tract, with muchgreater expression in the cecum and colon. Immunohistochem-istry experiments showed that the Duox2 proteins are localizedat the brush border of the enterocytes, indicating that Duox2 isexpressed in highly differentiated cells. The same results wereobtained with human colon. Furthermore, an NADPH-depen-dent, H2O2-generating activity was found to be linked toDuox2 protein expression in porcine tissues. The biochemicalcharacteristics of this H2O2 generator are the same as those ofthyroidal NADPH oxidase: it was solubilized under the sameconditions, and its calcium-dependent activity was similarlymodified by PAO treatment, which induced complete calciumdesensitization of the enzyme. These new data constitute fur-ther evidence suggesting that Duox2 is implicated in theproduction of H2O2 catalyzed by calcium-dependent NADPHoxidases. No H2O2 production was measurable in the humancolon, unlike in porcine tissues. Because H2O2 production
activity may be associated with digestive function, this differ-ence could be due to the physiological state of the humantissues, which were taken from patients who had been fastingfor 24 h, whereas the porcine tissues were obtained fromnormally fed animals. However, a species-related difference inDuox2 expression level and/or enzyme intrinsic activity cannotbe excluded. Indeed, we previously observed that the NADPHoxidase activity of particulate fractions from human thyroidwas 10–20 times lower than that of equivalent fractions frompig thyroid gland (21).
A discrepancy between the Northern and Western blot analysesfindings was observed. DUOX2 transcript levels were not relatedto protein expression. The Duox2 protein appeared to be wellexpressed in the digestive tract, but its mRNA level varied indifferent tissues, and in some of them, it could only be detected byRT-PCR. As the Northern blot experiments were carried out usingtotal RNA and not with polyA� mRNAs on the one hand, and aslong mRNAs transfer less efficiently than shorter ones on theother hand, DUOX2 mRNA was probably at the limit of detectionunder the experimental conditions used. Moreover, unlike whatwe had observed with porcine tissues, Northern blot analysis ofDUOX2 expression in human gastrointestinal tissues showed lowlevels of DUOX2 in the cecum and ascending colon and high
Fig. 8. Solubilization of the NADPH oxidase. A: distribution of the NADPH-dependent H2O2-generating activity after solubilization. Porcine colon partic-ulate fractions (F; 300 �l) were treated 1 h with the corresponding buffer andthen separated into the pellet (P) and supernatant (S) as described in MATERIALS
AND METHODS. Aliquots of all fractions (75 �l) were incubated under condi-tions described in MATERIALS AND METHODS with 0.4 mM EGTA, 0.5 mMCaCl2, and 0.1 mM NADPH. The results are representative of 2 independentexperiments. B: Western blot analysis of Duox solubilization. Aliquots of allfractions (20 �l) were analyzed by Western blot analysis as described inMATERIALS AND METHODS using the antibody raised again the Glu639-Arg1039
fragment of Duox2.
Fig. 7. Measurement of the NADPH-dependent H2O2-generating activities ingastrointestinal tissues. Particulate proteins from stomach (lane 1), duodenum(lane 2), ileum (lane 3), cecum (lane 4), sigmoidal colon (lane 5), floatingcolon (lane 6) and rectum (lane 7) were incubated under conditions describedin MATERIALS AND METHODS, with 0.1 mM NADPH and 0.4 mM EGTA in thepresence (filled bars) or absence (open bars) of 0.5 mM CaCl2. Similar datawere obtained from 2 additional experiments. The results are expressed asmeans � SE of 3 determinations.
G939DUAL OXIDASE2 EXPRESSED IN THE DIGESTIVE TRACT
expression in rectum (11). This difference could be ascribed to thephysiological state or fed state of the two species when the tissueswere taken.
So far, nonthyroid cell lines transfected with DUOX2 cDNAsalone have failed to generate H2O2 (6). The lack of H2O2
production could be related to the absence of the most glyco-sylated form of the Duox2 protein and/or to the absence ofDuox expression at the plasma membrane. Duox expression atthe plasma membrane was only obtained in the thyroid celllines, suggesting that an additional thyroid-specific componentis required to reconstitute a functional system. In contrast,Western blot analyses made with proteins from different di-gestive tract tissues revealed two proteins (165 and 175 kDa),corresponding to two different N-glycosylation states of theDuox2 protein previously detected in the thyroid. The compo-nent(s) required for the complete Duox2 maturation musttherefore also be expressed in the digestive tract.
The existence of two distinct DUOX genes raises the ques-tion of their respective roles. The expressions of their respec-tive mRNA are correlated in vivo in both normal thyroid (3)and in thyroid carcinomas (18) and are controlled in similarways by thyroid-stimulating hormone in vitro (5). It has beensuggested that they could be implicated in a heteropolymericstructure, with each subunit playing a distinct role in thecatalytic reaction. Our data showing that Duox1 is not ex-pressed at all, or only weakly expressed, in the digestive tractconsequently demonstrate that Duox2 is functionally indepen-dent of Duox1. In contrast, the DUOX1 gene appears to bepreferentially expressed along the mucosal surface of thetrachea and the bronchi (11).
Our findings in terms of proteins demonstrate that Duox2 isexpressed in highly differentiated colon epithelial cells and istherefore probably implicated in some function of these cells.Duox2 was found to be coexpressed with lactoperoxidase(LPO) in the salivary gland and rectum. It has been proposedthat Duox2 could be the source of H2O2 for LPO-catalyzedreactions and, therefore, could be implicated in a host-defensemechanism (11). The presence of Duox2 throughout the diges-tive tract, and particularly in the large intestine where themicrobial flora is most abundant, strengthens this hypothesis.
Interestingly, our data show for the first time that DUOX2expression is induced during the differentiation of the Caco-2cell line. This cell line therefore constitutes a good model forstudying the regulation of Duox2 expression in the colon.
NADPH oxidase 1 (NOX1) is another novel homolog ofgp91phox identified in the human colon and in the coloniccarcinoma cell line Caco-2 (27). NOX1 mRNA was recentlyfound to be abundantly expressed in the epithelial cells of themouse colon, with the greatest expression occurring predomi-nantly in the first two-thirds of the crypt (12). The NOX1transcript was induced in nonproliferating cells, suggesting thatNox1 performs specialized functions in the differentiated epi-thelial cells of the colon rather than having the mitogenicfunction previously proposed (27). It was found that the Nox1protein was constitutively expressed in surface mucous cells ofthe guinea pig colon, and in primary culture, these cellsspontaneously secreted superoxide anions, suggesting thatNox1 is constitutively active in this tissue (16). Cell-freeassays revealed that Nox1 cannot generate O2
per se (29).Substantial O2
generation was only achieved in the presenceof native neutrophil cytosol stimulated with PMA (29). p41Nox
and p51Nox, novel p47phox and p67phox homologs, were foundto be essential for Nox1 to achieve potent oxidase activity (16).It was speculated that Nox1 could provide an oxidative barrierto defend the host against intestinal pathogens (16). Stimula-tion of Toll-like receptor (TLR)4 in guinea pig gastric mucosalcells by Helicobacter pylori LPS upregulated the Nox1 activity(15). Intestinal epithelial cells, T84 cells, specifically re-sponded to rFliC and induced Nox1, although they wereinsensitive to LPS (16). It was speculated that stomach andcolon may use different TLR members to recognize respectivepathogenic microbes and activate Nox1.
Our findings show that Duox2 constitutes another source ofROS in the gut in addition to NOX1. Further studies are neededto identify the respective roles of the two oxidase systems inthe epithelial cells of the colon and to explore their respectiveresponse to different stimulators.
Fig. 9. Ca2� desensitization of NADPH-dependent H2O2 formation by PAO.A: generation of H2O2 as a function of phenylarsine oxide (PAO) concentra-tion. The cecum particulate fraction (125 �g/ml protein) was incubated for 10min at 4°C with increasing concentrations of PAO, as described in MATERIALS
AND METHODS. The particulate fraction was centrifuged and washed. ResidualH2O2-generating activity was measured with (�) or without (}) Ca2� � 100�M NADPH. B: Ca2� desensitization as a function of PAO concentration.Ca2� desensitization (%) � 100 � (activity measured in the absence ofCa2�)/(activity measured in the presence of Ca2�). Similar data were obtainedfrom 2 additional experiments. The results are expressed as means � SE of 3determinations.
G940 DUAL OXIDASE2 EXPRESSED IN THE DIGESTIVE TRACT
ROS are implicated in the pathogenesis of the mucosallesion in inflammatory bowel disease (IBD) (22), and theirlevels are elevated in the mucosa of patients as well as inexperimental models of inflammation (17). Oxidative damageis a key contributor to a loss of barrier integrity and injury.Oxidation, which leads to the inhibition of essential proteinfunction by inflammatory cells (neutrophils, macrophages, andlymphocytes), is a potential mechanism of tissue injury thatmay contribute to the pathogenesis of the disease. However,IBD is a disorder affecting all segments of the gut whereDuox2, an H2O2-generating system, was found to be ex-pressed. Disturbance of this system could therefore be impli-cated in the development of the inflammatory process. This
work provides a starting point for investigating Duox2 as a newcandidate mediator of physiopathological processes.
ACKNOWLEDGMENTS
We are indebted to Drs. Claudine Geffrotin and Silvia Vincent-Naulleaufrom Laboratoire Mixte CEA-INRA de Radiobiologie et d’Etude du Genomefor collaboration and for assistance in tissue collection.
Stanislas Morand is the recipient of a fellowship from the French Ministerede l’Education Nationale de la Recherche et de la Technologie.
Sedami Gnidehou is the recipient of a fellowship from the French Embassyin Benin.
GRANTS
This work was supported in part by Bonus Qualite Recherche 2002,University of Paris XI.
Fig. 10. Expression of DUOX2 in Caco-2 cells. A: timecourse of DUOX2 mRNA induction in postconfluentCaco-2 cells. Caco-2 cells were allowed to reach con-fluence and were then cultured for 10 days. Cells wereharvested at different time points. Total RNA was thenextracted and analyzed by Northern blot analysis. Bot-
tom shows the 28S and 18S ribosomal RNA bandsdetected by methylene blue staining. B: determinationof the region of the extracellular domain of Duox2recognized by MAbs. Four deletions in the extracellulardomain of Duox2 were created as described in MATERI-ALS AND METHODS and expressed in E. coli after incu-bating for 4 h with 1 mM isopropyl-�-D-thiogalactopy-ranoside. Bacterial culture (1 ml) was centrifuged(10,000 g, 30 s), and the pellet formed was then resus-pended in 1 ml of sample buffer. Aliquots (10 �l) wereanalyzed by Western blot analysis using MAbs at adilution of 1:1,000. C: immunoblot analysis of Duox1/2.Proteins (30 �g) from particulate fraction of HEK293cells stably transfected with human DUOX1 cDNA(lane 1) and human DUOX2 cDNA (lane 2), fromporcine thyroid plasma membrane (lane 3), and fromhuman thyroid particulate fraction (lane 4) were ana-lyzed by Western blot analysis with MAbs at a dilutionof 1:500. D: induction of Duox2 protein expression inCaco-2 cells. Proteins from particulate fraction ofCaco-2 cells, harvested at time points indicated, wereanalyzed by Western blot analysis with MAbs at adilution of 1:500. Antibody binding was revealed byECL (Amersham). E: measurements of the NADPH-dependent H2O2-generating activities in Caco-2 cells.Caco-2 cells were harvested at days 5 and 10 at post-confluence. Particulate fractions from Caco-2 cells wereincubated under conditions described in MATERIALS AND
METHODS, with 0.1 mM NADPH and 0.4 mM EGTA inthe presence (filled bars) or absence (open bars) of 0.5mM CaCl2. The results are expressed as means � SE of3 determinations. *Significantly different for the t-test ifP � 0.05.
G941DUAL OXIDASE2 EXPRESSED IN THE DIGESTIVE TRACT
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