TFF3-peptide increases transepithelial resistance in epithelial cells by modulating claudin-1 and -2 expression

peptides 27 (2006) 3383–3390

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TFF3-peptide increases transepithelial resistance in epithelial cells by modulating claudin-1 and -2 expression Dirk Meyer zum Bu¨schenfelde, Rudolf Tauber, Otmar Huber * Department of Laboratory Medicine and Pathobiochemistry, Charite´—Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany

article info

abstract

Article history:

TFF3 plays an important role in the protection and repair of the gastrointestinal mucosa. The

Received 6 March 2006

molecular mechanisms of TFF function, however, are still largely unknown. Increasing

Received in revised form

evidence indicates that apart from stabilizing mucosal mucins TFF3 induces cellular signals

29 August 2006

that modulate cell–cell junctions of epithelia. In transfected HT29/B6 and MDCK cells stably

Accepted 29 August 2006

expressing FLAG-tagged human TFF3 we have recently shown that TFF3 down-regulates E-

Published on line 2 October 2006

cadherin, impairs the function of adherens junctions and thus facilitates cell migration in wounded epithelial cell layers. Here we investigate TFF3-induced effects on the composition

Keywords:

and function of tight junctions in these cells. TFF3 increased the cellular level of tightening

Claudin

claudin-1 and decreased the amount of claudin-2 known to form cation-selective channels.

Occludin

Expression of ZO-1, ZO-2 and occludin was not altered. The change in claudin-1 and -2

TFF3

expression in TFF3-expressing HT29/B6 cells was accompanied by an increase in the

Tight junction

transepithelial resistance in confluent monolayers of these cells. These data suggest that

Epithelial barrier

TFF3 plays a role in the regulation of intestinal barrier function by altering the claudin composition within tight junctions thus decreasing paracellular permeability of the intest-

Abbreviations: AOX1, alcohol oxidase 1

inal mucosa. # 2006 Elsevier Inc. All rights reserved.

BCA, bicinchoninic acid CIP, calf intestinal phosphatase CLDN-1, claudin-1 CLDN-2, claudin-2 ERK, extracellular signal-regulated kinase JAM, junctional adhesion molecule OCLN, occludin PBS, phosphate-buffered saline PDZ, PSD95/Dlg/ZO-1 PVDF, polyvinylidene-difluoride RT-PCR, reverse transcriptionpolymerase chain reaction TER, transepithelial resistance TFF, trefoil factor family ZO-1, zonula occludens protein 1 ZO-2, zonula occludens protein 2

* Corresponding author at: Zentralinstitut fu¨r Laboratoriumsmedizin und Pathobiochemie, Charite´—Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany. Tel.: +49 30 8445 2525; fax: +49 30 8445 4152. E-mail address: [email protected] (O. Huber). 0196-9781/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2006.08.020

3384 1.

peptides 27 (2006) 3383–3390

Introduction

The trefoil factor family (TFF) comprises a group of three small peptides, TFF1, TFF2 and TFF3 that contain one (TFF1 and TFF3) or two (TFF2) TFF-domains forming highly conserved three-loop structural motifs. Under physiological conditions, TFFs are predominantly expressed in the gastrointestinal mucosa [11]. Meanwhile expression in multiple other mammalian tissues was reported [10]. Up-regulation of TFF-peptide expression has been observed in different types of disease, e.g. gastrointestinal ulceration, inflammatory bowel diseases (ulcerative colitis and Crohn’s disease) and malignant tumors [5,12,20]. TFF-peptides appear to play an important role in the protection, repair and healing of the gastrointestinal mucosa [9]. Mice over-expressing human TFF1 or rat TFF3 in the intestine exhibited increased resistance to intestinal damage and ulceration [17,22]. Conversely, mice with deleted TFF1 or TFF3 genes showed a higher susceptibility to gastrointestinal injury [14,18]. However, the functional effects and the molecular mechanisms of action of TFF-peptides currently are only partially understood [25]. For TFF1 a direct interaction with mucins of the van Willebrand factor type was reported which appears to mediate this protective effect [27]. Their stability against gastric acid and luminal proteolysis as well as their abundance in the gut lumen qualifies trefoil peptides to exert their function at the luminal–mucosal interface. Presumably, TFF-peptides preserve the integrity of the epithelial barrier by promoting the formation of a continuous gel of mucous glycoproteins on the mucosal surface. Previous studies indicate that TFF-peptides in addition modulate epithelial cell–cell contacts. Several reports have shown that TFF-peptides induce a down-regulation of the Ecadherin/catenin complexes in adherens junctions thus contributing to enhanced cell migration and epithelial repair [3,4,21]. The frequently observed functional interconnection between adherens junctions and tight junctions suggested that concomitant with changes in E-cadherin/catenin complexes, TFF-peptides also affect tight junctions. Tight junctions constitute barriers regulating the selective flux of ions and water between the compartments separated by epithelia and endothelia. Occludin and claudins together with junctional adhesion molecules (JAMs) represent the major transmembrane components of tight junctions. Occludin is a four-transmembrane protein with a molecular weight of 64 kDa. Claudins represent a family of more than 20 different four-transmembrane proteins with apparent molecular masses of approximately 22 kDa. The functional integrity of tight junctions furthermore depends on a set of cytoplasmatic proteins including the PDZ-domain proteins ZO-1, ZO-2, ZO3, kinases and phosphatases which are associated at the inner surface of the plasma membrane by binding to the cytoplasmic domains of occludin and the claudins [19,30]. Recently, several studies have indicated an influence of claudins on the regulation of paracellular permeability [30]. Over-expression of claudin-4 in MDCK II cells results in a selective decrease in Na+-ion-permeability without a significant effect on Cl permeability [29]. Furthermore, it has been shown that claudin-2 forms cation-selective channels in MDCK cells, and that claudin-2 expression in transfected MDCK I cells that physiologically lack endogenous claudin-2 and exhibit high

transepithelial resistance (TER) causes a >20-fold decrease in TER [1,6]. These observations suggest that the molecular composition of tight junction strands plays a central role for the regulation of the barrier function of epithelia. It was therefore the aim of the present study to analyze whether TFF3 in addition to the observed effects on adherens junctions modulates the function and composition of tight junctions. In stably hTFF3-transfected HT29/B6 and MDCK cells [21] expression of the tightening claudin-1 was increased and that of the channel forming claudin-2 was decreased. No differences were observed in the expression of occludin, ZO-1 and -2 proteins. The changes in claudin-1 and -2 expression were paralleled by an accelerated and increased establishment of the transepithelial resistance in confluent monolayers of FLAG-hTFF3 transfected HT29/B6 cells. Our results suggest that TFF3 decreases the paracellular epithelial permeability through selective regulation of claudin expression.

2.

Materials and methods

2.1.

Cell lines

Experiments were performed with the colon-carcinoma cell line HT29/B6 and Mardine–Darby canine kidney (MDCK) cells stably transfected with pFLAG-CMV3-hTFF3 or with vector alone as control [21]. Transfected MDCK cells were cultured in DMEM (PAA GmbH, Co¨lbe, Germany) supplemented with 10% (v/v) fetal calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin and 200 mg/ml G418 (Invitrogen Life Technologies, Karlsruhe, Germany) at 37 8C and 5% CO2. Transfected HT29/B6 cells were cultured in RPMI-medium (PAA GmbH, Co¨lbe, Germany) supplemented with 10% (v/v) fetal calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin and 200 mg/ml G418 (Invitrogen Life Technologies, Karlsruhe, Germany) at 37 8C and 5% CO2. For quantitative analyses cells were seeded in culture plates coated (15 min at 4 8C) with 0.1 mg/ml collagen A (Biochrom KG, Berlin, Germany) or on transwell filters. After thawing of clones and within a series of experiments expression of FLAG-hTFF3 by the different clones was repeatedly checked by immunoprecipitation from the cell culture supernatant.

2.2.

Reagents and antibodies

The rabbit polyclonal antibodies against occludin, ZO-1, ZO-2, claudin-1 and claudin-2 were obtained from Zymed (Zytomed, Berlin, Germany) and the mouse monoclonal antibodies against anti-occludin and anti-ZO-1 were from BD Transduction Laboratories (Heidelberg, Germany). The mouse monoclonal anti-b-actin and anti-FLAG-M2 antibodies were purchased from Abcam (Acris GmbH, Hiddenhausen, Germany) and Sigma (Taufkirchen, Germany), respectively. Horseradish peroxidase-labeled goat anti-mouse IgG antibody was obtained from Dianova (Hamburg, Germany), Alexa FluorTM594 goat anti-rabbit IgG from Molecular Probes (Invitrogen, Karlsruhe, Germany). CompleteTM-EDTA protease inhibitor mix was from Roche Diagnostics (Mannheim, Germany), benzonase was obtained from Merck (Darmstadt, Germany). Reagents for molecular biology were obtained from

peptides 27 (2006) 3383–3390

New England Biolabs (Frankfurt am Main, Germany) or Roche Diagnostics (Mannheim, Germany). Novel polyclonal anti-hTFF3 antibodies were generated by immunization of rabbits and guinea pigs with a hTFF3-IgG-Fc fusion protein. To generate this construct human TFF3 cDNA was amplified by PCR using the primer pair: forward, 50 -GCG GGA TCC GCC GCC ATG GCT GCC AGA GCG CTC-30 ; reverse, 50 TCT GCC GGG AAG CTT ACT TAC CTA CGA AGG TGC ATT CTG CTT C-30 . The PCR product was digested with BamHI and HindIII and cloned into the pCMV5-IgG (obtained from D. Vestweber, Mu¨nster, Germany) encoding the Fc-hinge region of human IgG containing IgG intronic sequences [23]. The TFF3-IgG encoding sequence was subsequently cloned into pcDNA6V5HisB using restriction enzymes EcoRI and XbaI. After transfection into CHO cells RNA was isolated and used as a template to amplify the hTFF3-IgG encoding cDNA with the One-Step RT-PCR kit (Qiagen, Hilden, Germany) using oligonuleotides: forward, 50 -TGC AGT CTC GAG AAA AGA GAG GCT GAG GAG TAC GTG GGC CTG-30 ; reverse, 50 -GGC GGA ATT CTC ATT TAC CCG GAG ACA G-30 . This PCR product no longer containing intronic sequences was again digested with XhoI and EcoRI and ligated into the Pichia pastoris expression vector pPIC9. All cloning steps were verified by sequencing. For electroporation into the P. pastoris strain GS115, pPIC9-hTFF3hIgG was digested with StuI and subsequently cells were plated on MD agar deficient in histidine. Expression of hTFF3-IgG was induced with methanol for 72 h and hTFF3-IgG was detected in the supernatant by SDS-PAGE. Protein was concentrated by ammonium sulfate precipitation. The pellet was resuspended in PBS and extensively dialyzed against PBS. In a next step the protein was further purified by protein A-sepharose chromatography and eluted with 0.1 M glycine pH 2.5. Purified hTFF3-IgG was used for immunization of rabbits and guinea pigs.

2.3. Western blotting experiments and immunofluorescence microscopy 2.3.1.

Western blotting

On collagen A-coated culture dishes, 1.0  106 cells were plated and grown for 4 days. Cells were lyzed in 400 ml 4 8C cold SDS–lysis buffer (50 mM Tris/HCl pH 6.8, 2% (w/v) SDS, CompleteTM-EDTA protease inhibitor mix and 250 U/ml benzonase) for 30 min at 4 8C. Insoluble material was removed by centrifugation (20,800  g, 10 min, 4 8C) and the supernatant was used in Western blot experiments. Protein concentrations were determined with the BCA protein assay reagent (Pierce, KMF, St. Augustin, Germany). For Western blotting protein (occludin, 1 mg; b-actin, 1 mg; ZO-1 and -2, 10 mg; claudin-1 2.5 mg; claudin-2, 10 mg) was separated by SDS-PAGE and transferred to PolyScreen PVDF membrane (Perkin–Elmer Life Sciences, Rodgau-Ju¨gesheim, Germany). Blocking and incubation with antibodies and detection were performed as described previously [2,31]. Quantification and data analysis was performed on a FujiFilm LAS-1000 imager with the Image Gauge Version 3.2 software.

2.3.2.

Immunofluorescence microscopy

On culture dishes containing collagen A-coated glass coverslides, 1.0  106 HT29/B6 and 0.5  106 MDCK cells, respec-

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tively, were grown for 4 days, washed with PBS and fixed immediately with ice-cold methanol for 10 min. Subsequently, the slides were washed in PBS, and after blocking with 0.1% (v/ v) goat serum in PBS for 30 min at room temperature, cells were incubated with the first antibody (either one of antioccludin antibody 0.25 mg/ml; anti-ZO-1 antibody 0.25 mg/ml; anti-claudin-1 and -2 antibody 0.5 mg/ml) in PBS for 30 min at room temperature. After three washes in PBS, cells were incubated with Alexa FluorTM594 goat anti-rabbit IgG (diluted 1:1000 in PBS) for further 30 min. After washing, coverslides were mounted with ProTaqs Mount Fluor (Biocyc GmbH & Co. KG, Luckenwalde, Germany) and analyzed on a confocal laserscanning microscope (LSM 510 META, Zeiss, Jena, Germany) with a Plan-Neofluar objective (with 40/1.3 oil and with 63/ 1.4 oil) at an excitation wavelength of 543 nm.

2.4.

RT- PCR analyses

On collagen A-coated culture dishes, 1.0  106 cells were plated and grown for 5 days. Total RNA was extracted using the NucleoSpin1 RNA II Kit (Macherey&Nagel, Du¨ren, Germany) and RT-PCR performed with 0.3 mg RNA using the OneStep RT-PCR kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions with the following oligonucleotides: hGAPDH forward, 50 -AGC AAT GC0 C TTC TGC ACC ACC AAC-30 ; reverse, 50 -CCG GAG GGG CCA TCC ACA GTC T-30 ; canine GAPDH forward, 50 -TGA CCT GCC GCC TGG AGA AA-30 ; reverse, 50 -GGC GTC GAA GGT GGA AGA GTG-30 ; human claudin1 forward, 50 -TCT AGT ATC CAG ACT CCA GCG-30 ; reverse, 50 GAC TCG CTC GGG CGC CCG CGC-30 ; canine claudin-1 forward, 50 -GTT GCC ACA GCA TGG TAT G-30 ; reverse, 50 -TCA CAC GTA CTC TTT CCC AC-30 ; human claudin-2 forward, 50 -CAT GAT GGT GAC ATC CAG TGC-30 ; reverse, 50 -CCT GGT CTC GGG CGC CCG CGC-30 ; canine claudin-2 forward, 50 -CCG ATA GCA TGA AGT TCG AG-30 ; reverse, 50 -CCT GGT CTC GGG CGC CCG CGC-30 .

2.5.

Measurement of transepithelial resistance

The transepithelial resistance (Rt; V cm2) of the HT29/B6 monolayers was determined in Ussing chambers specifically designed for insertion of Millicell filters [13]. Electrical measurements were performed with two fixed pairs of electrodes (STX-2, World Precision Instruments, USA) connected with an impedancemeter (Sorgenfrei, Department of Clinical Physiology, Charite´ Campus Benjamin Franklin). Rt was calculated from the voltage deflections caused by an external 10 mA, 21 Hz rectangular current. Depth of immersion and position of the filters were standardized mechanically. The temperature was maintained at 37 8C during the measurements by a temperature-controlled heating plate. Resistance values were corrected for the resistance of the empty filter and the bathing solution. Stably transfected HT29/B6 cells (1.0  106) were seeded on Millicell HA filters (effective membrane area 0.6 cm2). Three filters were placed together into one conventional culture dish (o.d. 60 mm) and filled with 10 ml of culture medium. Confluence of the monolayers was reached after 4 days and transepithelial resistance measurements were performed on days 4–8. The presented data are mean values of three independent experiments.

3386 3.

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Results

3.1. Altered claudin-1 and -2 expression levels in FLAGhTFF3-transfected epithelial cells To analyze the expression of tight junction proteins in FLAGhTFF3-transfected HT29/B6 and MDCK cells, stably expressing clones and vector-control clones were grown for 4 days and subsequently proteins were extracted with SDS–lysis buffer.

To confirm that the analyzed clones (HT29/B6 clone K3 and K17; MDCK clone K1, K2 and K4) express and secret hFLAGTFF3, the cell culture supernatant was analyzed with new polyclonal anti-hTFF3 antibodies generated in rabbits or guinea pig. These antibodies specifically detect hTFF3 but not hTFF1 or hTFF2 (Fig. 1A) and immunoprecipitate FLAGhTFF3 from the cell culture supernatants of both cell lines (Fig. 1B and C). These analyses show that HT29/B6 clone K3 expresses less FLAG-hTFF3 compared to clone K17. This is

Fig. 1 – Characterization of polyclonal anti-hTFF3 antibodies and detection of FLAG-hTFF3 in the supernatants of stably transfected HT29/B6 and MDCK cells. (A) Recombinant hTFF1 (lane 1), His6-TFF2 (lane 2) and His6-TFF3 (lane 3) were analyzed by Western blotting with the rabbit anti-hTFF3 and guinea pig anti-hTFF3 antisera. The antibodies specifically detect hTFF3 and show no cross-reactivity with hTFF1 and hTFF2. (B) Secreted FLAG-TFF3 protein was immunoprecipitated with either rabbit anti-hTFF3 or guinea pig anti-hTFF3 antibodies from cell culture supernatants of mock-transfected (clones M1, M2) and pFLAG-hTFF3-transfected (clones K3, K17) HT29/B6 cells and analyzed by Western blotting with monoclonal anti-FLAG M2 antibody. (C) Analysis of cell culture supernatants from mock-transfected (clones M1, M2, M3) and pFLAG-hTFF3-transfected (clones K1, K2, K4) MDCK cells by immunoprecipitation with the rabbit anti-hTFF3 antibody and by Western blotting with anti-FLAG M2 antibody confirms the secretion of FLAG-tagged hTFF3. (D) Secreted FLAGhTFF3 protein was immunoprecipitated with the rabbit anti-hTFF3 antibody from cell culture supernatants of mocktransfected (clones M1, M2) and pFLAG-hTFF3-transfected (clones K3, K17) HT29/B6 cells. Immunoprecipitates were analyzed by non-reducing SDS-PAGE and Western blotting with the anti-FLAG M2 antibody. Monomeric and dimeric forms of FLAG-hTFF3 were detectable (D).

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Fig. 2 – Expression of tight junction proteins in FLAG-hTFF3-transfected cells. Lysates of FLAG-hTFF3- and controltransfected HT29/B6 (A) or MDCK (B) cells were analyzed by Western blotting with anti-claudin-1 and -2 antibodies. The claudin-1 and -2 levels were quantified by chemiluminescence imaging on a FujiFilm LAS-1000 system. The signals for control-transfected HT29/B6 or MDCK cells were set to 100%. Data represent mean values W S.E.M. of three independent experiments. The levels of ZO-1, ZO-2 and occludin in lysates of FLAG-hTFF3- and control-transfected HT29/B6 (C) and MDCK (D) cells were detected with anti-ZO-1, anti-ZO-2 and anti-occludin antibodies. b-Actin was used as loading control. The presented figure is a representative of at least three independent experiments.

consistent with our previous observations showing that K3 exhibits slower migratory activity in wound filling assays and less prominent reduction of E-cadherin [21]. In non-reducing SDS-PAGE monomeric and dimeric forms of FLAG-hTFF3 were detectable (Fig. 1D). Expression of claudin-1, claudin-2, occludin, ZO-1 and ZO-2 was analyzed by Western blotting comparing two mocktransfected HT29/B6 clones with clones K3 and K17 and three mock-transfected MDCK clones with FLAG-hTFF3-expressing MDCK clones K1, K2 and K4, respectively. In both FLAG-hTFF3transfected HT29/B6 cell clones, in particular in clone K17 the claudin-2 protein level was strongly decreased as compared to control cells. In contrast claudin-1 expression was increased (Fig. 2A). These alterations in claudin-1 and -2 expression were also detectable in lysates of MDCK cell clones K1, K2 and K4 transfected with FLAG-tagged human TFF3 (Fig. 2B). No significant differences in the cellular levels of occludin, ZO1 or ZO-2 were detectable in FLAG-hTFF3-transfected and transfected HT29/B6 (Fig. 2C) and MDCK cells (Fig. 2D). Effects of hTFF3 on the cellular level of claudin-1 and -2 in FLAGhTFF3-transfected HT29/B6 and MDCK cells were confirmed by confocal immunofluorescence microscopy. In both FLAGhTFF3-transfected HT29/B6 (Fig. 3A) and MDCK (Fig. 3B) cells claudin-2 staining at cell–cell borders was reduced, whereas

claudin-1 staining was enhanced. The levels of ZO-1 and occludin were unchanged in both cell lines.

3.2. Transcriptional regulation of claudin-1 and -2 in FLAG-hTFF3-transfected epithelial cells To investigate whether the observed changes in claudin expression occur at the transcriptional level, RT-PCR experiments were performed. Increased amounts of claudin-1 mRNA were detectable in FLAG-hTFF3-transfected HT29/B6 cell clones K3 and K17 as compared to mock-transfected control cells. Down-regulation of claudin-2 mRNA levels was observed in the FLAG-hTFF3-transfected HT29/B6 cells (Fig. 4A). Similarly, in hFLAG-TFF3-transfected MDCK cells all clones revealed reduced levels of claudin-2 (shown for clones K2, an K4). In contrast to HT29/B6 cells only a minor increase in the level of claudin-1 was detectable (Fig. 4B).

3.3. Increased transepithelial resistance in FLAG-hTFF3transfected HT29/B6 cells The observed differences in the expression of the tightening claudin-1 and of the channel forming claudin-2 suggested that as a result tight junction function is altered

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Fig. 3 – Immunofluorescence microscopical detection of endogenous tight junction proteins. Cellular levels of claudin-1, claudin-2, ZO-1 and occludin in FLAG-hTFF3-transfected HT29/B6 (A) or MDCK (B) cells were analyzed by immunofluorescence microscopy with anti-claudin-1, anti-claudin-2, anti-ZO-1 and anti-occludin antibodies. Bar represents 10 mm. in FLAG-hTFF3-transfected cells compared to controls. We therefore measured the transepithelial resistance in mockand FLAG-hTFF3-transfected HT29/B6 cells. Four days after seeding on Transwell filters, FLAG-hTFF3- and mocktransfected cells formed confluent monolayers. During the following 4 days the TER of control cell layers remained unchanged, whereas TER in monolayers of FLAG-hTFF3transfected HT29/B6 cells increased by more than 2.5-fold (Fig. 5). TER was particularly increased in clone K17 in line with the higher amounts of FLAG-hTFF3 secreted into the cell culture supernatant and the more distinct alteration of claudin-1 and -2 expression in this cell clone as compared to clone K3.

4.

Fig. 4 – Changes in claudin-1 and -2 mRNA levels in FLAGhTFF3-transfected HT29/B6 and MDCK cells. RT-PCR experiments were performed with total RNA isolated from mock- and FLAG-hTFF3-transfected HT29/B6 (A) and MDCK (B) cells with primer pairs specific for claudin-1 and -2. GAPDH was amplified as a loading control. The figure is a representative of three independent experiments.

Discussion

TFF-peptides play an important role in the protection and repair of the gastrointestinal mucosa. The molecular mechanisms of TFF action are still not clear, but there is increasing evidence that functional and structural changes of apical junctional complexes are involved in TFF-induced modulation of gastrointestinal epithelia. In previous studies it was shown that components of the cadherin/catenin complex in adherens junctions are down-regulated in response to hTFF3 [3,4,21]. Using our previously established experimental system of HT29/B6 and MDCK cells stably transfected with FLAG-tagged human TFF3 [21] we here show that TFF3 causes an increase in

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Fig. 5 – Transepithelial resistance Rt in FLAG-hTFF3transfected HT29/B6 cells. Mock- or FLAG-hTFF3transfected HT29/B6 cells (clone K3 and K17) were seeded on Millicell-HA filters. Rt was monitored from days 4–8 after plating. The presented data are mean values W S.E.M. of three independent experiments.

the cellular level of claudin-1 and a decrease in claudin-2 as detected by Western blot analysis and immunofluorescence microscopy. In contrast to claudin-1 and -2, expression levels of the tight junction components occludin, ZO-1 and -2 were not affected by TFF3. There is increasing evidence that the composition of claudins within tight junction complexes contributes to the regulation of paracellular permeability [28]. We therefore hypothesized that the observed changes in claudin composition induced by TFF3 in HT29/B6 and MDCK cells might affect the TER of monolayers of these epithelia. Indeed, monitoring TER in monolayers of FLAG-hTFF3-transfected HT29/B6 cells showed a strong and time-dependent increase of the TER as compared to vector controls. This increase of TER in response to TFF3 is in line with flux studies showing that TFF3 induced by hypoxia-inducible factor 1 protects intestinal barrier function [7] and with an enhanced basal transepithelial resistance in the jejunum of transgenic mice expressing rTFF3 in jejunal tissue [17]. The presented data support the assumption that the effect of TFF3 in the protection of the mucosal barrier in part depends on an increase of epithelial resistance, i.e. on a decrease in paracellular permeability in response to alterations in the claudin composition of tight junctions induced by TFF3. The molecular mechanisms how TFF3 affects the protein composition of tight junctions and as a consequence paracellular permeability are yet unknown. The observation that TFF3 does not affect expression of occludin, ZO-1 and -2 indicates that the TFF3-dependent increase of TER does not correlate with a general increase in the amount of tight junction proteins, but is the result of the increased expression of the tightening claudin-1 and the decrease in the channel forming claudin-2. Thus our observations are consistent with previous studies showing that the expression of claudin-2 is inversely correlated with TER [1,6]. In summary, these data indicate that alterations of cellular claudin-1 and -2 levels contribute to the TFF3-mediated stabilization of intestinal epithelial barrier function. We cannot exclude, however, that

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also other claudins are modulated by TFF3 and contribute to the observed effect on TER. Multiplex RT-PCR revealed an increase of claudin-1 mRNA levels in FLAG-hTFF3-transfected HT29/B6 cells whereas claudin-2 mRNA was down-regulated. We therefore conclude that TFF3 influences tight junction protein composition at least in part at the level of claudin-1 and -2 transcription. The molecular mechanisms involved in the regulatory effects of TFF3 on transcription are unknown. Numerous studies suggest that tight junction permeability may be rapidly changed through cell signaling pathways that alter the composition of claudins within the tight junction complex. There is evidence that ERK1/2 is activated by TFF3 [8,16,24,26] and that ERK 1/2 may regulate the expression level of claudin2 in MDCK cells [15]. Whether an activation of ERK is involved in the regulation of claudin expression observed in the present study has to be examined in further studies. In summary, we here provide first evidence that TFF3 induces changes in the claudin composition of tight junctions contributing to the TFF3-mediated stabilization and maintenance of intestinal epithelial paracellular barrier function. These results extend previous studies that TFF3 induces multiple changes of cell–cell contacts contributing to the function of TFF3 in epithelial protection, migration and repair.

Acknowledgments This work was supported by the Stiftung fu¨r Pathobiochemie und Molekulare Diagnostik of the Deutsche Vereinte Gesellschaft fu¨r Klinische Chemie und Laboratoriumsmedizin (DGKL). We thank Prof. M. Fromm for gift of HT29/B6 cells and helpful discussions, Prof. D. Vestweber for the IgG-Fc vector, Barbara Kosel for expert technical assistance and Anja Fromm, Institute of Clinical Physiology of the Charite´, for help in TER measurements.

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