Loss of Smad5 leads to the disassembly of the apical junctional ...

1 downloads 0 Views 4MB Size Report
Jan 6, 2011 - liferation, migration, and apical junctional complex (AJC) protein expression were ..... directional cell motility in epithelial cells (20). Moreover,.
Am J Physiol Gastrointest Liver Physiol 300: G586 –G597, 2011. First published January 6, 2011; doi:10.1152/ajpgi.00041.2010.

Loss of Smad5 leads to the disassembly of the apical junctional complex and increased susceptibility to experimental colitis Joannie M. Allaire,1 Mathieu Darsigny,1 Sébastien S. Marcoux,1 Sébastien A. B. Roy,1 Jean-Francois Schmouth,1 Lieve Umans,2 An Zwijsen,2 François Boudreau,1 and Nathalie Perreault1 1

Faculté de Médecine et des Sciences de la Santé, Département d’Anatomie et Biologie Cellulaire, Université de Sherbrooke, Sherbrooke, Quebec, Canada; and 2VIB K.U. Leuven, Department of Molecular and Developmental Genetics, Leuven, Belgium Submitted 4 February 2010; accepted in final form 29 December 2010

Allaire JM, Darsigny M, Marcoux SS, Roy SAB, Schmouth JF, Umans L, Zwijsen A, Boudreau F, Perreault N. Loss of Smad5 leads to the disassembly of the apical junctional complex and increased susceptibility to experimental colitis. Am J Physiol Gastrointest Liver Physiol 300: G586 –G597, 2011. First published January 6, 2011; doi:10.1152/ajpgi.00041.2010.—The regulation of intestinal epithelial cell adhesion and migratory properties is often compromised in inflammatory bowel disease (IBD). Despite an increasing interest in bone morphogenetic protein (Bmp) signaling in gut pathologies, little is known of the specific roles played by individual Smads in intestinal epithelial functions. In the present study, we generated a mouse model with deletion of Smad5 transcriptional effector of the Bmp signaling pathway exclusively in the intestinal epithelium. Proliferation, migration, and apical junctional complex (AJC) protein expression were analyzed by immunofluorescence and Western blot. Human intestinal biopsies from control and IBD patients were analyzed for SMAD5 gene transcript expression by quantitative PCR (qPCR). Smad5⌬IEC and control mice were subjected to dextran sulfate sodium (DSS)-induced experimental colitis, and their clinical and histological symptoms were assessed. Loss of Smad5 led to intestinal epithelial hypermigration and deregulation of the expression of claudin-1 and claudin-2. E-cadherin was found to be equally expressed but displaced from the AJC to the cytoplasm in Smad5⌬IEC mice. Analysis of SMAD5 gene expression in human IBD patient samples revealed a significant downregulation of the gene transcript in Crohn’s disease and ulcerative colitis samples. Smad5⌬IEC mice exposed to experimental DSS colitis were significantly more susceptible to the disease and had impaired wound healing during the recovery phase. Our results support that Smad5 is partly responsible for mediating Bmp signals in intestinal epithelial cells. In addition, deficiency in epithelial Smad5 leads to the deregulation of cell migration by disassembling the AJC with increasing susceptibility to experimental colitis and impairment in wound healing. bone morphogenetic protein signaling; smad5 transcription factor; intestinal epithelial cell migration; inflammatory bowel diseases; apical junctional complex; wound healing THE INTESTINAL EPITHELIUM represents a dynamic system with rapid cell turnover (14, 21). The adult intestinal mucosa is composed of undifferentiated pluripotent stem cells, located in the lower portion of the intestinal crypt, and differentiated functional epithelial cells distributed along the villus axis. The intestinal stem cell undergoes asymmetrical division to produce one stem cell, which remains multipotent and undifferentiated, and a daughter cell that is committed to differentiate

Address for reprint requests and other correspondence: N. Perreault, Département d’Anatomie et Biologie Cellulaire, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, 3001 12e Ave. Nord, Sherbrooke, QC, Canada, J1H 5N4 (e-mail: [email protected]). G586

(6, 14, 52). This committed daughter cell goes through a number of cell divisions while migrating from the proliferative crypt to the differentiated villus compartment, where it finally exfoliates. This renewal process occurs over ⬃2–3 days in the mouse small intestine. Therefore, the coordination between proliferation, migration, differentiation, and apoptosis is central for the development, morphogenesis, and maintenance of intestinal epithelial architecture. The adhesive and migratory properties of epithelial cells are important for the establishment of the barrier function. Barrier integrity plays a critical role in normal development and is often compromised in a number of diseases, including inflammatory bowel disease (IBD) (7, 10). Tight junctions (TJ) and adherens junctions (AJ) regulate the barrier as well as cell-cell adhesion functions. Both TJ and AJ represent large complexes composed of transmembrane and cytosolic proteins (2, 25). The extracellular domain of the transmembrane proteins mediates homophilic interactions between adjacent cells, hence providing a mechanical link between the cell membranes that constitute the physical barrier (2, 25). The intracellular function of these transmembrane proteins is mediated through the interaction of the actin cytoskeleton with cytosolic proteins. These junctions are positioned on the cellular apical side of the lateral cell membrane to form the apical junctional complex (AJC). This complex is a highly dynamic entity influencing cell polarization, proliferation, differentiation, migration, and paracellular permeability (57). For example, dysregulation of TJ protein expression, such as claudins, leads to dramatic changes in intestinal paracellular permeability and ion exchange (51, 55, 60). Importance of the E-cadherin AJ component during intestinal epithelial cell adhesion and migration was first demonstrated by the breakthrough chimeric-transgenic experiments performed by Hermiston’s group (29, 30). Perturbed E-cadherin function in the crypt epithelium in this context was associated with increased cell migration, cell proliferation, and apoptosis. The epithelial barrier of these mice was also disrupted and acquired the morphological characteristics of the epithelium of Crohn’s disease (CD) patients (29). E-cadherin has also been shown to be increasingly detected in the cytoplasm of patients with IBD (36). Recently, polymorphisms in the E-cadherin gene were linked to its increased cytoplasmic accumulation in Crohn’s samples, suggesting that a subtle defect in E-cadherin function or localization may contribute to intestinal inflammatory susceptibility (47). These combined experiments therefore reveal that defects in AJC assembly can correlate with a loss of barrier function and cell-cell contact leading to the acquisition of migratory cell potential in adult gut tissues. Internalization of AJC proteins

0193-1857/11 Copyright © 2011 the American Physiological Society

http://www.ajpgi.org

Smad5 IN CELL MIGRATION AND EXPERIMENTAL COLITIS

appears to be a common mechanism to rapidly regulate epithelial barrier function and cell-cell adhesion allowing the remodeling of intercellular junctions (8, 33, 35, 47). However, the mechanisms by which AJC proteins are delocalized and endocytosed remain poorly understood (34). Bone morphogenetic proteins (Bmps) are multifunctional growth factors belonging to the transforming growth factor-␤ (TGF-␤) superfamily. Bmps play active roles in many developmental processes, in homeostasis as well as in various cellular functions in postnatal and adult animals (12, 43). Bmps signal through the serine/threonine kinase receptor subtypes I and II, where the type I receptor is activated upon Bmp-ligand binding and associates with the type II receptor. This activated receptor complex leads to the transphosphorylation of the Br-Smad proteins, which include Smad1, Smad5, and Smad8. These phosphorylated Br-Smads associate with the related protein Smad4 (Co-Smad), a shared partner of the TGF-␤ superfamily. The Br-Smad/Co-Smad complex then translocates to the nucleus where it activates transcription of specific target genes. Individual Br-Smads share a close homology (44) with a distinct pattern of expression (3, 11, 56). Smad1 and Smad5 function cooperatively in the early embryo (3). Phenotypic differences observed in knockout (KO) mice suggest a significant specificity for these factors in vivo (4, 19, 42, 56, 63). As with the majority of KO mice for Bmp pathway effectors, Smad1 and Smad5 KO embryos are not viable and die at embryonic day 10.5 (11). In addition to the defects documented in angiogenesis and in extraembryonic tissues, Smad5 KO mice develop a vestigial gut lacking a foregut pocket and entrance to the hindgut diverticulum (11). This observation suggests a significant role for Smad5 in early intestinal organogenesis. Significant advancements in the understanding of the in vivo function of Bmp ligands and their receptors in the development and maintenance of the digestive tract have been achieved in recent years (15, 24, 26, 31). However, the specific roles played by the various Br-Smads in intestinal homeostasis and cellular functions are still of limited knowledge. In the present study, we conditionally inactivated Smad5 in the mouse intestinal epithelium to delineate its function within the Bmp signaling cascade. Using this model, we uncovered an important role for Smad5 in intestinal epithelial cell migration. Our data indicate that loss of Smad5 promotes intestinal cell migration by disassembling the AJC through internalization of E-cadherin. We also demonstrate that the deficiency in epithelial Smad5 leads to increased susceptibility to IBD and impairment of wound healing. MATERIALS AND METHODS

Animals. C57BL/6-Smad5fx/fx mice were provided by Dr. A. Zwijsen (58) while the C57BL/6 12.4KbVilCre transgenic line was provided by Dr. D. L. Gumucio (40). Genomic DNA was isolated using the Spin Doctor genomic DNA kit from Gerard Biotech according to the manufacturer’s protocol. All mutations were genotyped using protocols already published (40, 58). All experiments were approved by the animal research committee of the Faculty of Medicine and Health Sciences of the Université de Sherbrooke. Tissue preparation and histological staining. Digestive tracts from 90- to 120-day-old control littermates and Smad5⌬IEC mice were fixed, sectioned, and stained [hematoxylin and eosin (H&E)] as previously described (5).

G587

Tissue collection, RNA extraction, and gene expression analysis. Total RNA was isolated and processed using the Totally RNA extraction kit (Ambion). Reverse-transcription PCR (RT-PCR) and quantitative real-time PCR were performed as described previously (5). PCR conditions and primer sequences are available upon request. Isolation of mouse adult intestinal epithelium. RNA was isolated from pure epithelial intestinal fractions of adult mice by an adaptation of the MatriSperse dissociation method described previously for human intestine (49). Briefly, mice were killed, and the intestine was separated in sections of jejunum. Each section was opened longitudinally and rinsed with cold PBS. The sections were further cut in 5-mm pieces and incubated in 5 ml of cold MatriSperse (Becton-Dickinson) in 15-ml tubes at 4°C for 18 –24 h. The epithelial layer was dissociated by gentle manual shaking. The epithelial suspension was collected, centrifuged, and washed with cold PBS. RNA extraction was performed as described above. Bromodeoxyuridine incorporation, transferase dUTP nick-end labeling assay, and immunofluorescence. Mice were injected with 10 ␮l of bromodeoxyuridine (BrdU; Zymed) per gram of body weight 90 min (for the proliferation assay), 12 h (for the colonic migration assay), or 48 h (for the jejunum migration assay) before death. For the apoptosis assay, the transferase dUTP nick-end labeling (TUNEL) assay was performed following the manufacturer’s protocol (Roche Diagnostics). Immunofluorescence staining was performed as previously described (5). The following antibodies were used at the indicated dilutions: anti-BrdU (AB no. BMC 9318, 1:50; Roche Diagnostics), E-cadherin (AB no. C20820, 1:1,000; BD Transduction Laboratories), ␤-catenin (AB no. 9587, 1:1,000; Cell Signaling), claudin-1 (AB no. 51–9000, 1:500; Invitrogen), claudin-2 (AB no. 51– 6100, 1:500; Invitrogen), FITC-conjugated anti-mouse IgG (1: 200; Vector), and FITC-conjugated anti-rabbit IgG (1:200; Vector). Protein extraction and western blot analysis. Total proteins were isolated from the intestinal mucosa of 90- to 120-day-old Smad5⌬IEC mice and control littermates with RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% Triton X-100, 1 mM EDTA, 0.2% SDS, and 0.5% sodium deoxycholate) containing protease and phosphatase inhibitors (9). Twenty-five micrograms of protein extract were analyzed by 10% BisTris NuPAGE (Invitrogen) and transferred to a polyvinylidene difluoride blotting membrane (Roche Diagnostics). Western blotting was then performed as previously described (50). The following affinity-purified antibodies were used: E-cadherin mouse monoclonal antibody (1:5,000) and claudin-1 (1:500), claudin-2 (1:1,000), and occludin (1:500) rabbit polyclonal antibodies from Zymed Laboratories (Invitrogen); cofilin (1:1,000), phosphorylated (p)-cofilin (1:1,000), and ␤-catenin (1:1,000) antibodies from Cell Signaling; and p-Smad5 (1:1,000) rabbit polyclonal antibodies from Abcam. For densitometry analyses, exposed films of Western blots were scanned, and images were analyzed using ImageJ (Rasband WS, ImageJ, United States National Institutes of Health, Bethesda, MD). CD and ulcerative colitis samples. TissueScan Real-Time Crohn’s and colitis disease panels were purchased from Origene Technologies. Each panel was composed of total isolated RNA from 6 control samples, 21 CD samples, and 21 ulcerative colitis (UC) samples collected from different individuals. After resuspension of lyophilized cDNAs, qRT-PCR was performed as described above and was normalized against PBGD. PCR conditions and primers used are available upon request. Induction and assessment of dextran sulfate sodium-induced colitis and recovery. Colitis was induced with dextran sulfate sodium (DSS) as previously described (48). Two independent groups were fed 3% (wt/vol) DSS water (4 controls and 4 Smad5⌬IEC/group) ad libitum for 2, 4, and 7 days (mol wt 35,000 –50,000; MP Biomedicals) as previously described (59). For water recovery experiments, two independent groups were fed 3% (wt/vol) DSS water (10 controls and 10 Smad5⌬IEC/group) ad libitum for 7 days followed by a recovery period of 5 days with water only. One group of CD-1 mice was fed 4%

AJP-Gastrointest Liver Physiol • VOL

300 • APRIL 2011 •

www.ajpgi.org

G588

Smad5 IN CELL MIGRATION AND EXPERIMENTAL COLITIS

(wt/vol) DSS water ad libitum for 2, 4, and 7 days. One group of Smad5⌬IEC mice compared with control mice and one group of 365-day-old Smad5⌬IEC mice compared with control mice were also used as water-only controls. Mice were killed on day 4 or 7 and assessed for disease activity on a 0 – 4 scale using the modified criteria from Cooper et al. (13). Scoring was as follows: weight loss (0, none; 1, 1–5%; 2, 6 –10%; 3, 11–20%; 4, 21% or more), stool consistency (0, solid; 2, loose and adhering to anus; 4, severe diarrhea, often with emptied colon), fecal blood (0, none; 2, lightly colored; 4, heavily colored), rectal bleeding (0, none; 2, moderate; 4, heavy), and colon length (0, 100 –96%; 1, 95– 86%; 2, 85–76%; 3, 75– 66%; 4, ⬍66%) compared with nontreated mice. Disease activity index (DAI) represented the total of all measured criteria. Histological grading of colitis. Colons were fixed in a Swiss-roll orientation in neutral buffered formalin followed by paraffin embedding. Histological scoring was compiled by two different individuals from 12 high-field images of H&E-stained sections taken blindly with a Leica DC300 camera on a DMLB2 microscope (Leica Microsystem Canada). Scoring, as validated by Dieleman et al. (16), was as

follows: severity of inflammation (0, none; 1, mild; 2, moderate; 3, severe), extent of inflammation (0, none; 1, mucosa; 2, mucosa and submucosa; 3, transmural), crypt damage (0, none; 1, first 1/3 of crypt base; 2, 2/3 of crypt base; 3, only surface epithelium remaining; 4, no epithelium remaining). All scores were added to represent the histological colitis score. Electron microscopy. Portions of mouse intestinal segments were rinsed with PBS, prefixed for 15 min with a 1:1 mixture of culture medium (Dulbecco’s modified Eagle’s medium) and freshly prepared 2.8% glutaraldehyde in cacodylate buffer (0.1 M cacodylate and 7.5% sucrose) and then fixed for 30 min with 2.8% glutaraldehyde at room temperature. After two rinses, specimens were postfixed for 1 h with 2% osmium tetroxide in cacodylate buffer. The tissues were then dehydrated using graded ethanol concentrations (40, 70, 90, 95, and 100%, three times each) and coated two times for 3 h with a thin layer of Araldite 502 resin (for ethanol substitution). Finally, the resin was allowed to polymerize at 60°C for 48 h. The specimens were detached from the plastic vessels, inverted in embedding molds, immersed in Araldite 502, and polymerized at 60°C for 48 h. Ultramicrotome-

Fig. 1. Impact of the loss of Smad5 on intestinal crypt-villus architecture. Quantification of Smad1, Smad5, and Smad8 in the intestinal epithelium was performed by quantitative PCR (qPCR) (n ⫽ 5). Intestinal epithelia of Smad5⌬IEC mice displayed a 97% decrease in Smad5 expression when compared with control littermates as opposed to no modulation in Smad1 and Smad8 expression (A) (Mann-Whitney U-test, ***P ⬍ 0.0001). Hematoxylin and eosin (H&E) staining was performed on paraffin sections of jejunum and colon from either 90-day-old control (B and D) or Smad5⌬IEC (C and E) mice. Normal intestinal and colonic mucosa was observed in controls (B and D). Smad5⌬IEC mice displayed abnormal intestinal morphology with expanded villi (C). The colonic gland of Smad5⌬IEC mice displayed no difference in morphology (E). The length of jejunal villi and crypts as well as colonic glands (F) was determined using MetaMorph software and H&E-stained micrographs of jejunum and colon (n ⫽ 4) (2-way ANOVA, ***P ⬍ 0.0001). Magnification ⫻20. Error bars represent SE.

AJP-Gastrointest Liver Physiol • VOL

300 • APRIL 2011 •

www.ajpgi.org

G589

Smad5 IN CELL MIGRATION AND EXPERIMENTAL COLITIS

prepared thin sections were contrasted with lead citrate and uranyl acetate and then observed on a Jeol 100 CX transmission electron microscope. All reagents were purchased from Electron Microscopy Sciences (Cedarlane, Hornby, ON, Canada).

Quantification and statistical analyses. All histological and cell count analyses or scores were performed using continuous sections from low-powered fields of well-oriented intestinal cross sections in a blind manner on an average of 10 independent fields/animal. The

Fig. 2. Increased migration but not proliferation and apoptosis in Smad5⌬IEC mice. Proliferation/migration assays by bromodeoxyuridine (BrdU) staining and apoptosis assays by transferase dUTP nick-end labeling (TUNEL) staining were performed on paraffin sections of jejunum and colon from either 90-day-old control (A, C, F, H, K, and M) or Smad5⌬IEC (B, D, G, I, L, and N) mice. Proliferation assays were performed by peritoneal BrdU injection, and animals were killed 90 min after injection. Proliferating cells stained by BrdU incorporation (green labeling) are found at the bottom of the crypts and colonic gland in Smad5⌬IEC (B and D, respectively) and control (A and C) mice. Evans blue dye served as a counterstain (red staining). BrdU-positive cells were counted from the jejunum and colon of control and Smad5⌬IEC mice (n ⫽ 4). Statistical analysis of the no. of positive BrdU cells revealed no modulation of proliferation in Smad5⌬IEC compared with control mice (E). Migration assays were performed by peritoneal BrdU injection, and animals were killed 48 h after injection for the jejunum or 12 h after injection for the colon. Note the increased speed of migration in the jejunum of Smad5⌬IEC mice (G) (white arrows) compared with controls (F). No significant modulation in migration rate was observed in colonic epithelium of Smad5⌬IEC mice (I) compared with controls (H). Evans blue dye served as a counterstain (red staining). Evaluation of epithelial cell migration was determined by measuring the distance end to end from the bottom of the crypt or gland to the last labeled migrating cell (green labeling) in the villus or upper gland over the total length of the crypt/villus axis in the jejunum or the entire gland in the colon in control and Smad5⌬IEC mice. The rate of migration was determined in BrdU-immunostained micrographs of jejunum and colon using MetaMorph software. This quantification revealed a significant increase in jejunal cell migration in Smad5⌬IEC mice but no modulation in corresponding colonic glands (J) compared with controls (n ⫽ 4). Apoptosis assays were performed by TUNEL immunostaining (green labeling) on sections from the small intestine and colon. No modulation in the levels of apoptosis was observed in either segment of Smad5⌬IEC mice (L and N) (white asterisk) compared with controls (K and M). DAPI staining served as a counterstain for visualization of the nucleus (blue staining). Statistical analysis of the no. of TUNEL-positive cells in the small intestine and colon of Smad5⌬IEC and control mice showed no significant difference in apoptosis (n ⫽ 4) (O) (2-way ANOVA, ***P ⬍ 0.0001). Magnification ⫻20 (F, G, K, and L) and ⫻40 (A-D, H, I, M, and N). Error bars represent SE.

AJP-Gastrointest Liver Physiol • VOL

300 • APRIL 2011 •

www.ajpgi.org

G590

Smad5 IN CELL MIGRATION AND EXPERIMENTAL COLITIS

numbers of BrdU-labeled cells were quantified as described previously (5, 37). Briefly, proliferation was measured by counting the number of BrdU-labeled cells per crypt. Villus and crypt morphometry as well as rate of migration were determined using the MetaMorph software (Universal Imaging). Images were imported, and magnification was calibrated by comparison with a stage micrometer (graticules; Tonbridge, Kent, UK). Statistical analysis was performed using two-way ANOVA. Differences were considered significant with a P value of ⬍0.05. For densitometry, qRT-PCR, and DAI score analyses, data were analyzed with the Student’s t-test for normal distribution data or using the Mann Whitney test for abnormal distribution. Two-way ANOVA test was used for statistical analysis of repeated measures. Differences were considered significant with a P value of ⬍0.05. All statistical analyses were carried out using Graph Pad Prism 5 (Graph Pad, San Diego, CA). RESULTS

Loss of epithelial intestinal Smad5 increases intestinal epithelial cell migration and length of the villus compartment. Homozygous floxed Smad5 mice (Smad5fx/fx) (58) were crossed with the villin-Cre transgenic line, which exclusively directs expression of the transgene in all intestinal epithelial cells, including stem cells (40). Conditional KO mice for

Smad5 (Smad5⌬IEC) were born at the expected Mendelian ratios and grew normally without obvious gross physical abnormalities. Because Smad5 is expressed in both epithelial and mesenchymal counterparts of the intestine, the nonenzymatic MatriSperse dissociation technique was used to separate the epithelium from the underlying mesenchyme (49) to evaluate epithelial expression levels of the Br-Smads in our mutant animals. Comparative analysis of Smad1, Smad5, and Smad8 mRNA expression by qPCR between control and Smad5⌬IEC mice confirmed a 97% epithelial loss of Smad5 expression in the intestine and colon with no compensatory changes in Smad1 or Smad8 expression in Smad5-deficient mice (Fig. 1A). Because the MatriSperse technique does not allow the exclusion of intraepithelial lymphocytes, this 3% residual expression is more likely to come from such cells still trapped in the epithelium. To assess morphological changes occurring with the loss of the downstream signal mediator Smad5, histological analysis was performed with H&E staining of the small intestine in control and Smad5⌬IEC mice. The intestine of Smad5⌬IEC mice displayed abnormal epithelial intestinal morphology with elongated villi (Fig. 1C) compared with control littermates (Fig.

Fig. 3. Ultrastructural evaluation of the apical junctional complex and immunolocalization of zonula occludens proteins in Smad5⌬IEC mice. Ultrastructural analysis exposed the presence of a less tight tight junction (TJ) and shallow adhesion plaques at the adherens junction (AJ) in Smad5⌬IEC mice in both jejunum (B) and colon (D) compared with controls (A and C, respectively). Immunostaining with a claudin-1 specific antibody showed that claudin-1 was diffused in the cytoplasm of jejunal and colonic epithelial cells in Smad5⌬IEC (F and H, respectively) compared with its localization at the apical membrane in control littermates (E and G, respectively). Immunostaining with a claudin-2 specific antibody showed that claudin-2 was localized normally at the apical membrane in both jejunal and colonic epithelial cells in Smad5⌬IEC (J) and in control (I) littermates. Claudin-2 expression was found to be higher in the colonic gland of Smad5⌬IEC mice (L) compared with controls (K). Magnification ⫻40,000 (A–D) and ⫻40 (E-K). D, desmosomes.

AJP-Gastrointest Liver Physiol • VOL

300 • APRIL 2011 •

www.ajpgi.org

Smad5 IN CELL MIGRATION AND EXPERIMENTAL COLITIS

1B); conversely, no difference in colonic gland morphology was observed in Smad5⌬IEC mice (Fig. 1E) compared with controls (Fig. 1D). Measurement of crypt and villus length in Smad5⌬IEC compared with control littermates revealed a significant 1.15-fold increase in jejunal villus length in Smad5⌬IEC compared with control (Fig. 1F) but no modulation in either jejunal crypt or colonic gland length in Smad5⌬IEC (Fig. 1F). Following the demonstration of an increase in villus lengthening in Smad5⌬IEC mice, we analyzed whether proliferation, migration, and/or apoptosis was altered in these mice. Proliferation assays with a 90-min BrdU pulse before death revealed that proliferation occurred in only a few cells at the bottom of the jejunal crypts and colonic gland of control mice (Fig. 2, A and C). No significant modification in the number or localization of proliferating cells was observed in either tissue in Smad5⌬IEC mice (Fig. 2, B and D). Statistical analysis of the number of positive BrdU cells in both tissues revealed no significant variation in proliferative cells between Smad5⌬IEC and control mice (Fig. 2E). Migration assays with a 48-h BrdU pulse before death showed an increase in epithelial cell migration rate along the jejunal crypt/villus axis in Smad5⌬IEC mice (Fig. 2G) compared with control littermates (Fig. 2F). Conversely, migration assays with a 12-h BrdU pulse before death revealed no modulation of the migration rate of colonic epithelial cells between Smad5⌬IEC (Fig. 2I) and control mice (Fig. 2H). Statistical analysis of the distance of epithelial migration in Smad5⌬IEC mice compared with control littermates showed a significant 1.22-fold increase in intestinal epithelial migration in Smad5⌬IEC mice but no modulation in colonic glands (Fig. 2J). Apoptosis assays performed by TUNEL immunostaining on small intestine and colon revealed no significant differences in the number of apoptotic cells in the epithelium of either tissue in Smad5⌬IEC mice (Fig. 2, L and N) compared with control (Fig. 2, K and M). Statistical analysis of the number of TUNEL-positive cells in both tissues corroborated the absence of modulation of apoptosis between Smad5⌬IEC and control mice (Fig. 2O). Deregulation of the epithelial AJC and dephosphorylation of cofilin in mice deficient for epithelial Smad5. Constant reorganization of TJ and AJ is a characteristic feature of processes involving epithelial cell migration and maintenance of epithelial barrier integrity in the gut (10, 33, 47, 57). These junctions are positioned on the apical side of the lateral cell membrane and form the AJC. To analyze the potential modification of the AJC in Smad5⌬IEC mice, ultrastructural analysis was performed using electron microscopy. Ultrastructural analysis exposed the presence of a less tight TJ and shallow adhesion plaques at the AJ in Smad5⌬IEC jejunum (Fig. 3B) and colon (Fig. 3D) comparatively to control mice (Fig. 3, A and C, respectively). Because it has been shown that alterations in expression or distribution of TJ proteins may impair the functionality of this junction (25, 51), immunostaining with occludin, claudin-1, claudin-2, and ZO-1 specific antibodies was performed on jejunal and colonic sections of control and mutant mice to analyze any modulation or mislocalization of proteins constituting the TJ. Analysis revealed a cytoplasmic relocalization for claudin-1 in Smad5⌬IEC (Fig. 3, F and H) compared with control mice (Fig. 3, E and G), whereas a modest increase in claudin-2 expression was observed in both the jejunum and colon of mutant mice (Fig. 3, J and L) compared with controls (Fig. 3, I and K). Moreover, claudin-2

G591

expression was found to be higher in the colonic gland of Smad5⌬IEC (Fig. 3L) compared with its normal expression in controls (Fig. 3K). Finally, no modulation or mislocalization was observed for occludin or ZO-1 (data not shown). The presence of shallow adhesion plaques at the AJ in Smad5⌬IEC jejunum (Fig. 3B) suggested the presence of either low levels of E-cadherin in Smad5⌬IEC mice or a mistargeting of the protein to the lateral membrane. To analyze the potential involvement of a mislocalization of E-cadherin at the lateral membrane of epithelial cells, immunostaining was performed

Fig. 4. Immunolocalization of zonula adherens proteins in Smad5⌬IEC mice. Immunostaining with an E-cadherin specific antibody showed that E-cadherin was diffused in the cytoplasm of jejunal and colonic epithelial cells in Smad5⌬IEC (B and D, respectively) compared with its localization at the lateral membrane in control littermates (A and C, respectively). Immunostaining with a ␤-catenin specific antibody showed that the E-cadherin cytoplasmic-associated protein ␤-catenin was diffused in the cytoplasm of jejunal and colonic epithelial cells in Smad5⌬IEC (F and H, respectively) compared with its localization at the lateral membrane in control littermates (E and G, respectively). Magnification ⫻40 (A–H).

AJP-Gastrointest Liver Physiol • VOL

300 • APRIL 2011 •

www.ajpgi.org

G592

Smad5 IN CELL MIGRATION AND EXPERIMENTAL COLITIS

on jejunal and colonic sections using an E-cadherin specific antibody. A cytoplasmic relocalization of E-cadherin was observed in Smad5⌬IEC mice (Fig. 4, B and D), whereas Ecadherin was found in the lateral membrane of control littermates (Fig. 4, A and C). Because ␤-catenin is known to be an E-cadherin cytoplasmic-associated protein, its localization was also investigated in our mutant mice by immunostaining with a ␤-catenin specific antibody on jejunal and colonic sections. Results also showed a moderate cytoplasmic relocalization for ␤-catenin in Smad5⌬IEC mice (Fig. 4, F and H), whereas ␤-catenin was found only in the lateral membrane in control animals (Fig. 4, E and G). To evaluate the expression level of these AJC proteins, Western blot analysis was performed on total extracts of jejunal and colonic mucosa. Results revealed a significant reduction in claudin-1 and an increase in claudin-2 expression levels in Smad5⌬IEC mice compared with controls in both jejunum (Fig. 5, A and B) and colon (Fig. 5, C and D). No modulation was observed however for claudin-3, claudin-4, claudin-8, or JAM-1 (data not shown). Occludin, E-cadherin, and ␤-catenin protein expression was not modulated by the loss of Smad5 in the intestinal epithelium (Fig. 5, A and B) and colonic mucosa (Fig. 5, C and D). The dynamic turnover of the actin cytoskeleton has previously been shown to play a role in AJC disassembly and cell

migration (34). Cofilin is an actin-binding factor required for the polymerization of actin filaments. In its phosphorylated form, cofilin is unable to bind actin, whereas dephosphorylation reactivates the actin-depolymerizing activity of cofilin (1). The dephosphorylation of cofilin has been shown to drive directional cell motility in epithelial cells (20). Moreover, dephosphorylation of cofilin and subsequent activation of the actin-depolymerizing activity can lead to the disruption of intracellular contact with the result that E-cadherin becomes rapidly internalized to accumulate in subapical cytosolic compartments (34). To investigate the phosphorylation status of cofilin-1 in Smad5⌬IEC mice, Western blot analysis was performed on total extracts of jejunal mucosa. A decrease in the phosphorylated status of cofilin-1 was observed in Smad5⌬IEC mice compared with controls, a tendency that was not reflected on the total cofilin-1 protein pool (Fig. 5, E and F). Smad5 gene transcript expression is decreased in IBD patients and during mouse experimental colitis. Because the integrity of the AJC if often altered during IBD, we next investigated if a deregulation in the expression of Smad5 could be associated with the disease. A gene expression analysis was performed for Smad5 in intestinal samples collected from both CD and UC patients. The levels of Smad5 gene transcript were significantly decreased by 44% in CD samples and by 67% in UC samples compared with controls (Fig. 6A). We next inves-

Fig. 5. Analysis of expression levels of apical junctional complex proteins and cofilin-1 phosphorylation status in mice impaired for epithelial Smad5. Western blot analysis of total jejunal (A) and colonic (C) mucosal lysates isolated from control or Smad5⌬IEC mice revealed a significant reduction in claudin-1 and an increase in claudin-2 expression levels in Smad5⌬IEC mice in both jejunum and colon (A and C, respectively). Occludin, E-cadherin, and ␤-catenin protein levels were not modulated by the loss of Smad5 in either jejunum or colon (A and C, respectively). Tubulin served as loading control for total protein extracts. Blots were scanned and analyzed with the ImageJ software, and values were normalized using the anti-tubulin scans (n ⫽ 10 for the jejunum and n ⫽ 5 for the colon) (B and D). Western blot analysis of total jejunal mucosa lysates isolated from control or Smad5⌬IEC mice revealed a decrease in phosphorylated cofilin-1 in Smad5⌬IEC compared with control mice (E). The total cofilin-1 protein pool was not modulated in Smad5⌬IEC mice compared with controls. Actin served as a loading control for total protein extracts. Blots were scanned and analyzed with the ImageJ software, and values were normalized using the anti-actin scans (n ⫽ 6) (F). Statistical significance of densitometry analyses was determined by Student’s t-test; *P ⬍ 0.05. Error bars represent SE.

AJP-Gastrointest Liver Physiol • VOL

300 • APRIL 2011 •

www.ajpgi.org

Smad5 IN CELL MIGRATION AND EXPERIMENTAL COLITIS

Fig. 6. Decreased expression of Smad5 in inflammatory bowel disease (IBD) patients and in dextran sulfate sodium (DSS)-treated mice. qPCR analysis revealed a decrease in Smad5 expression in both Crohn’s and ulcerative colitis patient samples (A). A 44% decrease in Smad5 mRNA expression levels was observed in Crohn’s patients and a 67% decrease in ulcerative colitis patients (n ⫽ 20) (Mann-Whitney U-test; **P ⬍ 0.005). Smad5 mRNA expression was investigated in wild-type murine intestine treated with DSS to induce colitis (B). qPCR analysis revealed a decrease in Smad5 expression by 1.96-fold after 2 days and by 3.23-fold after 7 days of DSS treatment compared with untreated mice (B) (n ⫽ 4) (Student’s t-test; *P ⬍ 0.05 and **P ⬍ 0.005). Western blot analysis showed a decrease in Smad5 protein expression in mice treated with DSS for 2 and 4 days. This decrease was transient as demonstrated by the return to normal expression after 7 days of treatment (C). Error bars represent SE.

tigated whether Smad5 expression was modulated during DSSinduced colitis (48). As assessed by qPCR, Smad5 expression was decreased by 1.96-fold after 2 days and by 3.23-fold after 7 days of DSS treatment compared with untreated mice (Fig. 6B). Corroborating these data, Western blot analysis confirmed a similar reduction pattern of Smad5 protein in DSS-treated mice compared with untreated mice (Fig. 6C). However, reexpression of Smad5 protein was observed at 7 days of DSS treatment. Loss of intestinal epithelial Smad5 increases susceptibility to experimental colitis. The severity of DSS-induced colitis was next assessed in Smad5⌬IEC compared with control mice. Cohorts of Smad5⌬IEC and control mice were provided with either water only or water containing 3% (wt/vol) DSS for 7 days (Fig. 7, A–H). Body weight change, stool consistency, and the presence of fecal blood were recorded and used to calculate a clinical score that reflected the overall disease activity index. Smad5⌬IEC mice showed a more severe susceptibility (P ⬍ 0.05) to DSS-induced colitis at 4 days of treatment compared with control mice. By 7 days of treatment, Smad5⌬IEC mice showed a DAI of 9.33 compared with 5.43 for control mice (Fig. 7I). Histological damage to the colonic mucosal glandular

G593

architecture was more severe in Smad5⌬IEC mice (Fig. 7F) compared with control littermates (Fig. 7E). The severity of mucosal injury assessed microscopically revealed a histological score of 6.35 for Smad5⌬IEC compared with 4.48 for control mice and correlated with the clinical score of the DAI (Fig. 7J). Analyses of proliferation, migration, and apoptosis in the colon of DSS-treated mice revealed no significant modulation of these cell functions in mutant mice during DSS treatment (data not shown). To evaluate a possible role for Smad5 in the wound-healing processes, a DSS-water recovery treatment was performed where mutant and control mice were provided with water containing 3% (wt/vol) DSS for 7 days followed by a 5-day recovery phase with water only. At the outset, the DSS water recovery experiments in Smad5⌬IEC mice led to a 33% death rate during the 5-day water recovery phase (Fig. 7K). Analysis of the surviving animals following the completion of the recovery cycle showed a DAI of 2.4 for Smad5⌬IEC mice compared with 0.83 for controls (Fig. 7I). Histological analysis revealed that distal colon of mutant mice had not started to heal as observed by the presence of important areas denuded of epithelium (Fig. 7H) compared with controls (Fig. 7G). The severity of the mucosal injury was also confirmed by a significant histological score of 5.78 for Smad5⌬IEC compared with 4.90 for control mice (Fig. 7J) after 5 days of water recovery. Analysis of weight loss during the experiment demonstrated that Smad5⌬IEC mice were not inclined to regain the weight during the recovery phase (Fig. 7K). DISCUSSION

In recent years, the Bmp signaling pathway has been shown to play key crucial roles in gut morphogenesis, cell fate, and adult homeostasis (5, 24, 26). The wide range of biological responses obtained by Bmps is dependent on three downstream effectors, Smad1, Smad5, and Smad8. Despite increasing interest in gut Bmp signaling, very little is known of the specific roles played by individual Smads in intestinal epithelial cell functions and homeostasis. Herein, we revealed key roles for epithelial Smad5 within the Bmp signaling pathway in intestinal epithelial cell migration and AJC assembly. Of note, we observed a significant decrease in the level of Smad5 mRNA in samples from IBD patients and found that loss of Smad5 expression in the intestinal epithelium led to increased susceptibility to DSS-induced colitis in mice. We and others have previously reported that loss of the upstream receptor bone morphogenetic protein receptor type IA (BmpR1a) leads to an increased lengthening of crypts and villi as well as an increase in the number of epithelial proliferating cells and crypt units per villus (5, 26). In the present study, we show that loss of intestinal epithelial Smad5, one of the downstream effectors of the Bmp signaling pathway, led to a lengthening of the villi without any significant deregulation of crypt/villus architecture or the colonic gland. The loss of Smad5 also led to a deregulation of epithelial cell migration rate along the crypt/villus axis but did not alter epithelial cell proliferation. Epithelial migration rate in the colon on the other hand was not affected. This is in contrast to the BmpR1a epithelial KO phenotype where both cellular functions were found to be affected (5). These observations suggest that other Smad family members are likely involved or compensated for during Bmp-dependent epithelial cell proliferation.

AJP-Gastrointest Liver Physiol • VOL

300 • APRIL 2011 •

www.ajpgi.org

G594

Smad5 IN CELL MIGRATION AND EXPERIMENTAL COLITIS

The lengthening of the villi in the absence of either increased cell production in the crypt or decreased apoptosis as seen in Smad5⌬IEC could be explained by the increased migration rate of epithelial cells along the crypt/villus axis without an accelerated extrusion rate of these cells from the apex of the villus (28). Therefore, based on the differential phenotypes observed between the upstream receptor BmpR1a (5) and the downstream effector Smad5, our findings indicate that epithelial

Smad5 is crucial for Bmp-dependent regulation of intestinal epithelial cell migration but not for proliferation or apoptosis. This observation thus excludes Smad5 as a possible contributor to the repopulating of the epithelial cell pool following insult or injury to the intestinal epithelial sheet even if Bmp signaling is known to be involved in intestinal epithelial cell proliferation (5, 26). To our knowledge, this is the first report showing that

AJP-Gastrointest Liver Physiol • VOL

300 • APRIL 2011 •

www.ajpgi.org

Smad5 IN CELL MIGRATION AND EXPERIMENTAL COLITIS

Smad5 is responsible for mediating some of the specific Bmp signals targeting intestinal epithelial cells. The AJC consisting of the TJ and the AJ plays an active role during cell migration, polarization, and in the maintenance of epithelial barrier integrity (22, 23, 28, 30, 33, 57). The constant reorganization of these two junctional entities is a characteristic feature of processes involving epithelial cell migration in the gut (35). Moreover, due to its critical effects on epithelial intestinal cell migration, E-cadherin expression and localization have been shown to be tightly regulated in normal epithelial cells (22, 23, 28, 29, 36). Our analysis revealed that loss of Smad5 did not affect E-cadherin protein or mRNA expression but did lead to changes in relocalization of the protein from the lateral membrane to the cytoplasm of the cell. Interestingly, ␤-catenin, the intracellular partner of E-cadherin, followed the same localization pattern in mutant mice with no increase in its expression levels. This relocalization of E-cadherin/␤-catenin suggests a possible defect in AJC assembly that could hypothetically lead to a loss of barrier function of cell-cell contact leading to the acquisition of migratory phenotypes. Of note, previous studies with intestinal epithelial cells have established a link between cytoskeleton turnover, AJC disassembly, and cell migration (27, 33–35). During this phenomenon, actin depolymerization factor (ADF)/cofilin-1 plays an active role in F-actin polymerization. In its phosphorylated form, ADF/cofilin-1 is unable to bind to actin, whereas its dephosphorylation can reactivate the actin-depolymerizing activity of ADF/cofilin (1). Dephosphorylation of ADF/cofilin is regulated by the slingshot (SSH) phosphatase and its phosphorylation by the LIM-kinase (32). Subsequently, depolymerization of the actin cytoskeleton leads to the disruption of intracellular contact and causes E-cadherin to become rapidly internalized (34). Our study revealed that Smad5⌬IEC mice harbored a reduction in their intestinal phosphorylated ADF/cofilin-1 pool, suggesting an implication for a deregulation of actin depolymerization in E-cadherin internalization in Smad5⌬IEC mice. This observation could also explain in part the increased migration rate seen in the intestinal epithelium of Smad5⌬IEC mice. Moreover, this observation supports a mechanism explaining the delocalization of E-cadherin into the cytoplasm of intestinal epithelial cells in the absence of Smad5. The basal level of ADF/cofilin-1 was not affected in Smad5⌬IEC mice, thus supporting that this target was not transcriptionally dependent on Smad5. Nonetheless, it can be implied that the loss of Smad5 affects the dynamic turnover of the actin cytoskeleton through a deregulation of the phosphorylated status of ADF/cofilin-1, thus causing increased internalization of E-cadherin to the cyto-

G595

plasm and leading to the disruption of the AJC and acquisition of migratory phenotypes. Similarly, activation of the Bmp signaling pathway by Bmp7 in culture neurons from Xenopus laevis growth cones has been shown to lead to decreased phosphorylation of ADF/cofilin-1 without a change in total ADF/cofilin protein thereby leading to the repulsive response of the Xenopus growth cones (62). In this latter study, Bmp7treated cultures showed a marked attraction for the growth cones instead of the repulsion after introduction of a DN-SSH phosphatase. Whether Smad5 leads to transcriptional repression of SSH-phosphatase or transcriptional activation of the LIM-kinase in intestinal epithelial cells remains to be determined. Another important role for AJC consists in regulating gut permeability, which is dependent on the coordinated expression and interaction of proteins in the TJ (60, 61). Indeed, it has been shown that TJ proteins show alterations in distribution or expression in IBD (10). For example, claudin-1 protein has been reported to be decreased in IBD. In contrast to claudin-1, claudin-2 expression is increased in colonocytes of patients with active IBD (51). In addition to its role with the AJC, our study revealed that the colonic epithelium of Smad5⌬IEC mice exhibited a reduction in claudin-1 and an increase in claudin-2 expression levels, thus suggesting an implication for Smad5 in the functionality of the TJ. Altogether, these observations strongly support a role for Smad5 in regulating the localization or expression of various AJC proteins and consequently participate in junctional functions such as gut permeability. Damage and impairment of the intestinal epithelium can be observed following acute insult or in the presence of various diseases. Under normal conditions, the intestinal epithelium rapidly reestablishes itself by three successive mechanisms: restitution (migration and dedifferentiation of the epithelial cells), proliferation, and, finally, maturation and differentiation of the epithelial cells (17, 54). In chronic inflammation, disruption at various stages of this wound-healing process is often observed. Over the years, involvement of the TGF-␤ signaling pathway in IBD pathogenesis and the wound-healing process has been widely studied (18, 45, 46, 54). For example, prophylactic or therapeutic Bmp-7 treatment in TNBS-treated rats led to protection against inflammation and resulted in accelerated wound healing (41). However, the relationship between IBD and specific elements of Bmp signaling, such as Smad5, has not been reported. Our data suggest that Smad5 could play an essential role in regulating important components of the AJC and possibly in maintaining mucosal integrity of the intestine by controlling epithelial barrier permeability. Another

Fig. 7. Increased susceptibility to experimental colitis and impaired wound healing during recovery in Smad5⌬IEC mice. For susceptibility experiments, Smad5⌬IEC mice were more affected than controls at 4 and 7 days following 3% DSS treatment, with a disease activity index (DAI) of 1.79 (4 days) and 9.67 (7 days) for Smad5⌬IEC compared with 0.71 (4 days) and 5.75 (7 days) for control mice (I) (n ⫽ 8). Histological analysis on DSS-induced colitis mice revealed greater histological damage to the colonic mucosa in Smad5⌬IEC after 7 days of treatment (F) compared with controls (E). H&E-stained colon sections from p90and p365-day-old Smad5⌬IEC (B and D, respectively) and control (A and C, respectively) mice at the beginning of treatment served as normal histological controls. Histological grading of colitis showed a histological score of 3.67 (4 days) and 6.35 (7 days) for Smad5⌬IEC compared with 2.31 (4 days) and 4.48 (7 days) for control mice (J) (n ⫽ 4). For recovery experiments, after a 3% DSS treatment for 7 days followed by a 5-day recovery phase with water only, Smad5⌬IEC mice showed a DAI of 2.4 compared with 0.83 for controls (I) (n ⫽ 20). Analysis of weight loss during the experiment demonstrated that Smad5⌬IEC mice were not inclined to regain the weight during the recovery phase (K). Note that the DSS water recovery experiments led to a 33% death rate in the Smad5⌬IEC population during the 5-day water recovery phase (K). Histological analysis following water recovery revealed that the distal colon of mutant mice had not begun to heal as observed by the presence of important areas denuded of epithelium (H) compared with controls (G). Histological grading of colitis following recovery showed a histological score of 5.78 for Smad5⌬IEC compared with 4.90 for control mice (J) (n ⫽ 10). Error bars represent SE (2-way ANOVA: *P ⬍ 0.05, **P ⬍ 0.005, and ***P ⬍ 0.0001). †Animals that died during the experiment. Magnification ⫻20 (A–H). AJP-Gastrointest Liver Physiol • VOL

300 • APRIL 2011 •

www.ajpgi.org

G596

Smad5 IN CELL MIGRATION AND EXPERIMENTAL COLITIS

possibility may be that Smad5 influences the wound-healing process directly. The reexpression of Smad5 protein observed at 7 days in DSS-treated mice suggests a possible role for Smad5 in the restitution phase following injury. Indeed, loss of Smad5 could contribute to uncontrolled migration, hence inappropriately reestablishing the continuity of the surface epithelium after an extensive destruction. Under normal conditions, the redistribution of E-cadherin is not sufficient to influence intestinal epithelial polarization. However, following an inflammatory stress, the cytoplasmic redistribution of Ecadherin interferes with the normal function of the protein at the lateral membrane and affects the epithelial repolarization and differentiation phase of the wound-healing process, leading to a nonfunctional epithelium (47). In support of this, DSS water recovery experiments performed herein in Smad5⌬IEC mice led to a 33% death rate during the 5-day water recovery phase. Moreover, analysis of the surviving animals, after completion of the recovery cycle, revealed that mutant mice had not even begun to heal following the DSS insult, as observed by the presence of important areas denuded of epithelium in the colon. Because Smad5 is expressed in both epithelial and mesenchymal counterparts of the intestine, it remains plausible that the reduction in Smad5 expression in samples from IBD patients may not only be restricted to the epithelial component. However, by taking advantage of genetic manipulation in mice to delete Smad5 exclusively in the epithelial compartment, we were able to assess its specific contribution during IBD pathogenesis. Specific epithelial deletion of Smad5 caused an increased susceptibility to DSS colitis, demonstrating that epithelial Smad5 is protective during gut inflammation. Globally, there are very few studies on the transcriptional regulating roles of Smad5 (38, 39, 53). One of the future challenges will be to define Smad5 gene targets during the intestinal inflammatory response. In summary, the present data are the first to demonstrate that Smad5 is responsible for mediating some of the specific Bmp signals targeted at intestinal epithelial cells. More importantly, results demonstrate that the loss of Smad5 exclusively in the intestinal epithelial compartment promotes intestinal cell migration by disassembling the AJC through internalization of E-cadherin. This observed deficiency in epithelial Smad5 contributes to increased susceptibility to experimental colitis and impaired wound healing. Consequently, enhancing Bmp signaling through Smad5 may represent an attractive strategy for a protective role of this effector during IBD. ACKNOWLEDGMENTS We thank M. P. Garand and S. Lamarre for help with statistical analysis, the QTRN/CToDE intestinal phenotyping platform from the Université de Sherbrooke for their histology and phenotyping services, Denis Martel and Charles Bertrand for assistance with electron microscopy analysis, and Dr. D. L. Gumucio for providing the 12.4KbVilCre transgenic line. GRANTS This research was supported by the Canadian Institutes of Health Research (MOP-84327 to N. Perreault) and a grant from the Crohn’s and Colitis Foundation of Canada. N. Perreault and F. Boudreau are members of the FRSQ-funded Centre de Recherche Clinique Étienne LeBel. F. Boudreau is a scholar from the Fonds de la Recherche en Santé du Québec.

DISCLOSURES The authors make the statement that there is no conflict of interest to disclose. REFERENCES 1. Agnew BJ, Minamide LS, Bamburg JR. Reactivation of phosphorylated actin depolymerizing factor and identification of the regulatory site. J Biol Chem 270: 17582–17587, 1995. 2. Anderson JM, Van Itallie CM, Fanning AS. Setting up a selective barrier at the apical junction complex. Curr Opin Cell Biol 16: 140 –145, 2004. 3. Arnold SJ, Maretto S, Islam A, Bikoff EK, Robertson EJ. Dosedependent Smad1, Smad5 and Smad8 signaling in the early mouse embryo. Dev Biol 296: 104 –118, 2006. 4. Aubin J, Davy A, Soriano P. In vivo convergence of BMP and MAPK signaling pathways: impact of differential Smad1 phosphorylation on development and homeostasis. Genes Dev 18: 1482–1494, 2004. 5. Auclair BA, Benoit YD, Rivard N, Mishina Y, Perreault N. Bone morphogenetic protein signaling is essential for terminal differentiation of the intestinal secretory cell lineage. Gastroenterology 133: 887–896, 2007. 6. Bach SP, Renehan AG, Potten CS. Stem cells: the intestinal stem cell as a paradigm. Carcinogenesis 21: 469 –476, 2000. 7. Berkes J, Viswanathan VK, Savkovic SD, Hecht G. Intestinal epithelial responses to enteric pathogens: effects on the tight junction barrier, ion transport, and inflammation. Gut 52: 439 –451, 2003. 8. Birchmeier W, Behrens J, Weidner KM, Hulsken J, Birchmeier C. Epithelial differentiation and the control of metastasis in carcinomas. Curr Top Microbiol Immunol 213: 117–135, 1996. 9. Boudreau F, Rings EH, van Wering HM, Kim RK, Swain GP, Krasinski SD, Moffett J, Grand RJ, Suh ER, Traber PG. Hepatocyte nuclear factor-1 alpha, GATA-4, and caudal related homeodomain protein Cdx2 interact functionally to modulate intestinal gene transcription. Implication for the developmental regulation of the sucrase-isomaltase gene. J Biol Chem 277: 31909 –31917, 2002. 10. Bruewer M, Samarin S, Nusrat A. Inflammatory bowel disease and the apical junctional complex. Ann NY Acad Sci 1072: 242–252, 2006. 11. Chang H, Huylebroeck D, Verschueren K, Guo Q, Matzuk MM, Zwijsen A. Smad5 knockout mice die at mid-gestation due to multiple embryonic and extraembryonic defects. Development 126: 1631–1642, 1999. 12. Chen D, Zhao M, Mundy GR. Bone morphogenetic proteins. Growth Factors 22: 233–241, 2004. 13. Cooper HS, Murthy SN, Shah RS, Sedergran DJ. Clinicopathologic study of dextran sulfate sodium experimental murine colitis. Lab Invest 69: 238 –249, 1993. 14. Crosnier C, Stamataki D, Lewis J. Organizing cell renewal in the intestine: stem cells, signals and combinatorial control. Nat Rev Genet 7: 349 –359, 2006. 15. De Santa Barbara P, Williams J, Goldstein AM, Doyle AM, Nielsen C, Winfield S, Faure S, Roberts DJ. Bone morphogenetic protein signaling pathway plays multiple roles during gastrointestinal tract development. Dev Dyn 234: 312–322, 2005. 16. Dieleman LA, Elson CO, Tennyson GS, Beagley KW. Kinetics of cytokine expression during healing of acute colitis in mice. Am J Physiol Gastrointest Liver Physiol 271: G130 –G136, 1996. 17. Dignass AU. Mechanisms and modulation of intestinal epithelial repair. Inflammatory bowel diseases 7: 68 –77, 2001. 18. Fiocchi C. TGF-beta/Smad signaling defects in inflammatory bowel disease: mechanisms and possible novel therapies for chronic inflammation. J Clin Invest 108: 523–526, 2001. 19. Flanders KC, Kim ES, Roberts AB. Immunohistochemical expression of Smads 1– 6 in the 15-day gestation mouse embryo: signaling by BMPs and TGF-betas. Dev Dyn 220: 141–154, 2001. 20. Ghosh M, Song X, Mouneimne G, Sidani M, Lawrence DS, Condeelis JS. Cofilin promotes actin polymerization and defines the direction of cell motility. Science 304: 743–746, 2004. 21. Gordon JI, Hermiston ML. Differentiation and self-renewal in the mouse gastrointestinal epithelium. Curr Opin Cell Biol 6: 795–803, 1994. 22. Gumbiner BM. Regulation of cadherin adhesive activity. J Cell Biol 148: 399 –404, 2000. 23. Gumbiner BM. Regulation of cadherin-mediated adhesion in morphogenesis. Nat Rev 6: 622–634, 2005.

AJP-Gastrointest Liver Physiol • VOL

300 • APRIL 2011 •

www.ajpgi.org

Smad5 IN CELL MIGRATION AND EXPERIMENTAL COLITIS 24. Haramis AP, Begthel H, van den Born M, van Es J, Jonkheer S, Offerhaus GJ, Clevers H. De novo crypt formation and juvenile polyposis on BMP inhibition in mouse intestine. Science 303: 1684 –1686, 2004. 25. Hartsock A, Nelson WJ. Adherens and tight junctions: structure, function and connections to the actin cytoskeleton. Biochim Biophys Acta 1778: 660 –669, 2008. 26. He XC, Zhang J, Tong WG, Tawfik O, Ross J, Scoville DH, Tian Q, Zeng X, He X, Wiedemann LM, Mishina Y, Li L. BMP signaling inhibits intestinal stem cell self-renewal through suppression of Wnt-betacatenin signaling. Nat Genet 36: 1117–1121, 2004. 27. Heath JP. Epithelial cell migration in the intestine. Cell Biol Int 20: 139 –146, 1996. 28. Hermiston ML, Gordon JI. In vivo analysis of cadherin function in the mouse intestinal epithelium: essential roles in adhesion, maintenance of differentiation, and regulation of programmed cell death. J Cell Biol 129: 489 –506, 1995. 29. Hermiston ML, Gordon JI. Inflammatory bowel disease and adenomas in mice expressing a dominant negative N-cadherin. Science 270: 1203– 1207, 1995. 30. Hermiston ML, Wong MH, Gordon JI. Forced expression of E-cadherin in the mouse intestinal epithelium slows cell migration and provides evidence for nonautonomous regulation of cell fate in a self-renewing system. Genes Dev 10: 985–996, 1996. 31. Howe JR, Sayed MG, Ahmed AF, Ringold J, Larsen-Haidle J, Merg A, Mitros FA, Vaccaro CA, Petersen GM, Giardiello FM, Tinley ST, Aaltonen LA, Lynch HT. The prevalence of MADH4 and BMPR1A mutations in juvenile polyposis and absence of BMPR2, BMPR1B, and ACVR1 mutations. J Med Genet 41: 484 –491, 2004. 32. Huang TY, DerMardirossian C, Bokoch GM. Cofilin phosphatases and regulation of actin dynamics. Curr Opin Cell Biol 18: 26 –31, 2006. 33. Ivanov AI, Hunt D, Utech M, Nusrat A, Parkos CA. Differential roles for actin polymerization and a myosin II motor in assembly of the epithelial apical junctional complex. Mol Biol Cell 16: 2636 –2650, 2005. 34. Ivanov AI, McCall IC, Parkos CA, Nusrat A. Role for actin filament turnover and a myosin II motor in cytoskeleton-driven disassembly of the epithelial apical junctional complex. Mol Biol Cell 15: 2639 –2651, 2004. 35. Ivanov AI, Nusrat A, Parkos CA. Endocytosis of the apical junctional complex: mechanisms and possible roles in regulation of epithelial barriers. Bioessays 27: 356 –365, 2005. 36. Karayiannakis AJ, Syrigos KN, Efstathiou J, Valizadeh A, Noda M, Playford RJ, Kmiot W, Pignatelli M. Expression of catenins and E-cadherin during epithelial restitution in inflammatory bowel disease. J Pathol 185: 413–418, 1998. 37. Langlois MJ, Roy SA, Auclair BA, Jones C, Boudreau F, Carrier JC, Rivard N, Perreault N. Epithelial phosphatase and tensin homolog regulates intestinal architecture and secretory cell commitment and acts as a modifier gene in neoplasia. FASEB J 23: 1835–1844, 2009. 38. Lee KS, Kim HJ, Li QL, Chi XZ, Ueta C, Komori T, Wozney JM, Kim EG, Choi JY, Ryoo HM, Bae SC. Runx2 is a common target of transforming growth factor beta1 and bone morphogenetic protein 2, and cooperation between Runx2 and Smad5 induces osteoblast-specific gene expression in the pluripotent mesenchymal precursor cell line C2C12. Mol Cell Biol 20: 8783–8792, 2000. 39. Lohmann F, Bieker JJ. Activation of Eklf expression during hematopoiesis by Gata2 and Smad5 prior to erythroid commitment. Development 135: 2071–2082, 2008. 40. Madison BB, Dunbar L, Qiao XT, Braunstein K, Braunstein E, Gumucio DL. Cis elements of the villin gene control expression in restricted domains of the vertical (crypt) and horizontal (duodenum, cecum) axes of the intestine. J Biol Chem 277: 33275–33283, 2002. 41. Maric I, Poljak L, Zoricic S, Bobinac D, Bosukonda D, Sampath KT, Vukicevic S. Bone morphogenetic protein-7 reduces the severity of colon tissue damage and accelerates the healing of inflammatory bowel disease in rats. J Cell Physiol 196: 258 –264, 2003. 42. Massague J. TGF-beta signal transduction. Annu Rev Biochem 67: 753– 791, 1998.

G597

43. Mishina Y. Function of bone morphogenetic protein signaling during mouse development. Front Biosci 8: d855–869, 2003. 44. Miyazawa K, Shinozaki M, Hara T, Furuya T, Miyazono K. Two major Smad pathways in TGF-beta superfamily signalling. Genes Cells 7: 1191–1204, 2002. 45. Monteleone G, Boirivant M, Pallone F, MacDonald TT. TGF-beta1 and Smad7 in the regulation of IBD. Mucosal Immunol 1, Suppl 1: S50 –S53, 2008. 46. Monteleone G, Kumberova A, Croft NM, McKenzie C, Steer HW, MacDonald TT. Blocking Smad7 restores TGF-beta1 signaling in chronic inflammatory bowel disease. J Clin Invest 108: 601–609, 2001. 47. Muise AM, Walters TD, Glowacka WK, Griffiths AM, Ngan BY, Lan H, Xu W, Silverberg MS, Rotin D. Polymorphisms in E-cadherin (CDH1) result in a mis-localised cytoplasmic protein that is associated with Crohn’s disease. Gut 58: 1121–1127, 2009. 48. Okayasu I, Hatakeyama S, Yamada M, Ohkusa T, Inagaki Y, Nakaya R. A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice. Gastroenterology 98: 694 –702, 1990. 49. Perreault N, Beaulieu JF. Primary cultures of fully differentiated and pure human intestinal epithelial cells. Exp Cell Res 245: 34 –42, 1998. 50. Perreault N, Katz JP, Sackett SD, Kaestner KH. Foxl1 controls the Wnt/beta-catenin pathway by modulating the expression of proteoglycans in the gut. J Biol Chem 276: 43328 –43333, 2001. 51. Prasad S, Mingrino R, Kaukinen K, Hayes KL, Powell RM, MacDonald TT, Collins JE. Inflammatory processes have differential effects on claudins 2, 3 and 4 in colonic epithelial cells. Lab Invest 85: 1139 –1162, 2005. 52. Scoville DH, Sato T, He XC, Li L. Current view: intestinal stem cells and signaling. Gastroenterology 134: 849 –864, 2008. 53. Singh R, Horsthuis T, Farin HF, Grieskamp T, Norden J, Petry M, Wakker V, Moorman AF, Christoffels VM, Kispert A. Tbx20 interacts with smads to confine tbx2 expression to the atrioventricular canal. Circ Res 105: 442–452, 2009. 54. Sturm A, Dignass AU. Epithelial restitution and wound healing in inflammatory bowel disease. World J Gastroenterol 14: 348 –353, 2008. 55. Tamura A, Kitano Y, Hata M, Katsuno T, Moriwaki K, Sasaki H, Hayashi H, Suzuki Y, Noda T, Furuse M, Tsukita S, Tsukita S. Megaintestine in claudin-15-deficient mice. Gastroenterology 134: 523– 534, 2008. 56. Tremblay KD, Dunn NR, Robertson EJ. Mouse embryos lacking Smad1 signals display defects in extra-embryonic tissues and germ cell formation. Development 128: 3609 –3621, 2001. 57. Turner JR. ‘Putting the squeeze’ on the tight junction: understanding cytoskeletal regulation. Semin Cell Dev Biol 11: 301–308, 2000. 58. Umans L, Vermeire L, Francis A, Chang H, Huylebroeck D, Zwijsen A. Generation of a floxed allele of Smad5 for cre-mediated conditional knockout in the mouse. Genesis 37: 5–11, 2003. 59. Vowinkel T, Kalogeris TJ, Mori M, Krieglstein CF, Granger DN. Impact of dextran sulfate sodium load on the severity of inflammation in experimental colitis. Dig Dis Sci 49: 556 –564, 2004. 60. Weber CR, Nalle SC, Tretiakova M, Rubin DT, Turner JR. Claudin-1 and claudin-2 expression is elevated in inflammatory bowel disease and may contribute to early neoplastic transformation. Lab Invest 88: 1110 – 1120, 2008. 61. Weber CR, Turner JR. Inflammatory bowel disease: is it really just another break in the wall? Gut 56: 6 –8, 2007. 62. Wen Z, Han L, Bamburg JR, Shim S, Ming GL, Zheng JQ. BMP gradients steer nerve growth cones by a balancing act of LIM kinase and Slingshot phosphatase on ADF/cofilin. J Cell Biol 178: 107–119, 2007. 63. Zwijsen A, van Grunsven LA, Bosman EA, Collart C, Nelles L, Umans L, Van de Putte T, Wuytens G, Huylebroeck D, Verschueren K. Transforming growth factor beta signalling in vitro and in vivo: activin ligand-receptor interaction, Smad5 in vasculogenesis, and repression of target genes by the deltaEF1/ZEB-related SIP1 in the vertebrate embryo. Mol Cell Endocrinol 180: 13–24, 2001.

AJP-Gastrointest Liver Physiol • VOL

300 • APRIL 2011 •

www.ajpgi.org