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Oxazolone-induced over-expression of focal adhesion kinase in colonic epithelial cells of colitis mouse model
FEBS Letters 584 (2010) 3949–3954
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Oxazolone-induced over-expression of focal adhesion kinase in colonic epithelial cells of colitis mouse model Yanju Ma a, Shingo Semba a, Atsuo Maemoto b, Masayuki Takeuchi a, Isamu Kameshita c, Atsuhiko Ishida d, Sanae Kato a, Tsuyoshi Katoh a, Yunpeng Liu e, Takanobu Taniguchi a,* a
Department of Biochemistry, Asahikawa Medical University, Asahikawa 078-8510, Japan Department of Internal Medicine, Asahikawa Medical University, Asahikawa 078-8510, Japan Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan d Laboratory of Molecular Brain Science, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan e Department of Medical Oncology, The First Hospital of China Medical University, Shenyang 110001, China b c
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Article history: Received 4 June 2010 Revised 9 July 2010 Accepted 27 July 2010 Available online 1 August 2010 Edited by Lukas Huber Keywords: Inflammatory bowel disease Ulcerative colitis Crohn’s disease Protein tyrosine kinase Focal adhesion kinase
a b s t r a c t We examined the change of protein tyrosine kinases (PTKs) expression levels in colonic epithelial cells isolated from mice in which colitis was induced by oxazolone administration, using the monoclonal antibody YK34, which cross-reacts with a wide variety of PTKs. We identified focal adhesion kinase (FAK) and found the expression level increased due to the induction of colitis. Furthermore, we found that there was a positive correlation between FAK expression and the severity of colitis. Also, FAK expression localized in the colonic epithelium but not in the lamina propria, implying FAK functions in epithelial cells during colitis formation and/or wound repairing. Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Ulcerative colitis and Crohn’s disease are known as inflammatory bowel diseases (IBD), which are characterized by the destruction of the gastrointestinal tract integrity, the excessive production of cytokines, and subsequent chronic intestinal inflammation. The dysregulation of the mucosal immune system was thought to play an important role during the initiation of inflammation [1]. In Crohn’s disease, T helper cell type 17 (Th17) cytokine interleukin-17 (IL-17), and Th1 cytokines interferon-c and TNF-a increased [2,3]. In ulcerative colitis, IL-17 and Th2 cytokines usually increased [2–4]. Therefore, the over-activation of immune cells in intestinal mucosa could cause chronic intestinal inflammation. Although IBD is thought to be related to genetic alterations [5] and environmental factors [6,7], their etiology and pathologic mechanisms remain largely unclear. In the mucosal immune system, the intestinal epithelium has important functions: as a physical barrier separating luminal bacteria and immune cells, and as an immunological barrier Abbreviations: IBD, inflammatory bowel disease; PTK, protein tyrosine kinase; FAK, focal adhesion kinase * Corresponding author. Fax: +81 166 68 2349. E-mail address: [email protected] (T. Taniguchi).
producing antimicrobial peptides and cytokines. The goblet cells secrete a layer of mucus that protect the intestinal epithelial cells from bacteria [8]. Disruption of epithelial integrity by dysregulation of the NF-jB pathway causes spontaneous chronic inflammation because of translocation of bacteria into mucosa [9]. Antimicrobial peptide expression by epithelial cells controls the microflora homeostasis [10,11]. Thus, the reduction of its expression is thought to be one of the causes of IBD [12]. It is also known that Toll-like receptors (TLRs), which are activated by intestinal bacteria and play a major role in the pathogenesis of IBD, are upor down-regulated in intestinal epithelial cells [13,14]. It has been believed that protein tyrosine kinases (PTKs) have critical roles for many cellular functions including immune response, ulcer formation and wound healing in the intestinal epithelium. In a previous study, Sugiyama et al. produced a monoclonal antibody, YK34, which cross-reacted with a wide variety of PTKs [15]. They used antigenic peptides corresponding to a highly conserved region among various PTKs to produce YK34. In addition, they detected the change of the PTK expression during differentiation of a leukemia HL60 cell by Western blot using YK34. We hypothesized that the PTKs expression changed with the dysregulation of intestinal epithelial functions caused by inflammation. We therefore induced chemical colitis in mice colons by intrarectal
0014-5793/$36.00 Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2010.07.054
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administration of oxazolone and examined the change of immunoreactive proteins with YK34 in colonic epithelial cell lysate by twodimensional gel electrophoresis (2D-GE) and Western blot. A database search using a determined pI value and the molecular weight of immuno-reactive spots identified focal adhesion kinase (FAK) as one of the PTKs in which the expression level was correlated with the severity of colitis. We also found that FAK was localized in epithelial cells regardless of the colitis induction. Our results suggest that FAK is involved in colitis formation and/or colonic epithelial inflammation. 2. Materials and methods 2.1. Materials 4-Ethoxymethylene-2-phenyl-2-oxazolin-5-one (oxazolone) was purchased from Sigma Chemical (St. Louis, MO, USA). Oxazolone was dissolved in 50% ethanol to obtain a 1% (w/v) solution. YK34 was raised against a highly conserved region (subdomain VIB) of PTKs [15]. Anti-FAK and -Fer antibodies were purchased from Abcam (Cambridge, UK). Anti-Syk and -b-actin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase-conjugated goat anti-rabbit antibody was purchased from Pierce (Rockford, IL, USA). 2.2. Induction of oxazolone colitis We induced colitis in Male BALB/c mice (weighing 20–25 g, which were bred in the Animal Care Facility of Asahikawa Medical University) by intrarectal administration of 100 ll of oxazolone solution via catheter delivered 4 cm into the colon [16]. Twentyfour hours after the administration, all the colonic tissues were dissected and used for further experiments. The degree of inflammation was assessed by macroscopic observation as described previously [17] using the following criteria: 0 – no inflammation; 1 – swelling or redness; 2 – swelling and redness; 3 – one or two ulcers; 4 – more than two ulcers or one large ulcer; 5 – mild necrosis; 6 – severe necrosis. All animal procedures were approved by the Animal Care Committees of Asahikawa Medical University.
7 M urea, 2 M thiourea, 5 mM Tris–HCl, 0.5% Nonidet P-40 and 5 mM DTT. Isoelectric focusing (IEF) was carried out using agarose IEF gels (AgarGEL, pH 3–8, ATTO, Tokyo, Japan) in the first dimension and 7.5% polyacrylamide slab gels in the second dimension according the manufacturer’s guidelines. 2.5. Western blot Proteins that were separated by gel electrophoresis were transferred to a Immobilon-P PVDF membrane (Millipore, Billerica, MA, USA). The membrane was blocked with Tris-buffered saline containing 0.05% Tween 20 (TBST) and 2% ECL Advance Blocking Reagent (GE Healthcare UK Ltd., Buckinghamshire, England). After incubation with the appropriate primary antibody, bound antibody was detected by using peroxidase-conjugated secondary antibody and ECL Advance Western Blotting Detection Kit (GE Healthcare). 2.6. Immunohistochemistry Colonic tissues dissected from mice were fixed with 20% buffered formalin and embedded in paraffin, and cut into tissue sections. After deparaffinization, each section was incubated with 1:100 dilution of anti-FAK antibody for 1 h at room temperature and then washed three times with PBS. Bound primary antibody was detected by using anti-rabbit antibody and diaminobenzidine staining. 2.7. Analysis of data The level of FAK protein expression was measured by densitometric measurement of the immuno-reactive signal with antiFAK antibody of the Western blots using ImageJ software. FAK expression levels were normalized based on b-actin expression levels in same blots. Results are expressed as the means ± S.E.M. of n experiments. Statistical significance was evaluated using Student’s t-test. P < 0.05 was considered statistically significant. The correlation between the clinical score and FAK expression level was evaluated by using Pearson product-moment correlation coefficient.
2.3. Preparation of colonic epithelial cells and its lysate 3. Results We prepared colonic epithelial cells as described [18], in which the purity of the colonic epithelial cell preparation has been shown to be approximately 90% [19]. Briefly, the dissected colon was opened by cutting longitudinally and gently rinsed with ice-cold phosphate-buffered saline (PBS). Then the colon was cut into pieces of about 5 mm 5 mm and transferred into a 2.0-ml microtube containing 1.5 ml of PBS. The tube was vortexed for 5 min at room temperature. After removal of the supernatant, 1 ml of PBS containing 20 mM EDTA was added into the tube. After vortexing for 10 min at room temperature, the cell suspension was collected by centrifugation. This step was repeated 6–8 times. Next, the cells were suspended in 33% Percoll and centrifuged for 20 min at 400g. After centrifugation, the cells on the top of the 33% Percoll were collected and used as epithelial cells. Then, the cells were suspended in five volumes of lysis buffer containing 0.5 M NaCl, 0.2 mM EDTA, 1.5 mM MgCl2, 1 mM DTT, 1 mM PMSF and 20 mM Tris–HCl, pH 7.5 and lysed by passing through a syringe equipped 22-gauge needle repeatedly. After centrifugation at 12 000g for 20 min at 4 °C, the lysate was used for the following experiments.
3.1. Induction of colitis by the administration of oxazolone We employed the oxazolone-induced colitis model that was accepted as a T helper-2 type model [20,21] similar to human ulcerative colitis. Since we found that the mortality of the oxazoloneadministered mice by the end of day 2 was approximately 50%, we examined the severity of colitis at 24 h posttreatment. At this time point, some oxazolone-administered mice showed soft stool, rectal bleeding and fecal staining around the anus. Macroscopic examination revealed that inflammation had occurred in the distal colon. However, the severity varied widely; about 30% of the oxazolone-administered mice showed no colitis, redness or swelling of the bowel wall (clinical score 0 and 1), whereas 40% exhibited mild to moderate inflammation (clinical score 2–4) and the other 30% displayed large ulcers accompanied with necrosis (clinical score 5 and 6). 3.2. Detection of immuno-reactive proteins with YK34 antibody in colonic epithelial cell lysate
2.4. Two-dimensional gel electrophoresis Cell lysates were desalted by trichloroacetic acid precipitation and the precipitates were dissolved in a sample buffer containing
In order to investigate the change of the PTK expression level, we carried out 2D-GE and Western blot analysis using the monoclonal antibody YK34. Twenty-four hours after the administration
Y. Ma et al. / FEBS Letters 584 (2010) 3949–3954
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of oxazolone, the epithelial cell lysate was prepared from each colonic tissue and analyzed by Western blot. As shown in Fig. 1, more than 10 immuno-reactive spots were detected. Among them, we found that the intensity of the spot, of which the molecular mass was 120 kDa and pI value was 6.3, was significantly and reproducibly increased by the administration of oxazolone (Fig. 1, arrowheads). On the basis of these values, we searched the UniProtKB/ Swiss-Prot database using the TagIdent program [22] with the following: tyrosine-protein kinases of Mus musculus with a molecular mass 120 000 ± 12 000 Da and pI value between 6.0 and 6.5. Several PTKs including FAK 1 were suggested by the search. 3.3. Oxazolone administration enhances FAK expression in epithelial cells Previous studies showed that FAK contributes to wound healing in gastric injuries [23,24] and FAK expression increased in epithelial cells near human gastric and colonic ulcers [25]. We thus examined the protein expression level of FAK in the colon epithelium. Lysates were prepared from colonic epithelial cells in mice and were subjected to Western blot using anti-FAK antibody. As shown in Fig. 2A, the expression level of FAK after oxazolone administration was significantly higher in all lysates prepared from inflamed colonic tissues compared with those from control tissues. In contrast, other PTKs such as Syk and Fer did not show any change of their expression levels. Densitometric analysis revealed that FAK expression in oxazoloneadministered mice increased to 168 ± 15.5% compared with ethanol-administered mice (P = 0.011, Fig. 2B). 3.4. FAK expression level correlates with severity of oxazolone-induced colitis Using Western blot analysis, we found the variance in the expression level of FAK in inflamed colonic epithelial cell lysates. In order to examine the correlation between the severity of colitis and FAK expression level, we assessed the degree of inflammation of each oxazolone-administered mouse colon. The estimated clinical score was plotted as a function of the relative expression level of FAK (Fig. 3). The correlation between the clinical score and FAK expression was modest (r = 0.681).
Fig. 2. Changes of the protein expression levels of FAK in epithelial cell lysates. (A) Representative results of Western blot. Lysates that were prepared from the colonic epithelial cells of control mice (lanes marked as ) or oxazolone-administered mice (lanes marked as +) were probed with anti-FAK, -Syk and -Fer antibodies in Western blot. The b-actin was also detected as a loading control. (B) Quantification of FAK expression level. Optical density of each band was measured by densitometry. The expression levels of FAK were then normalized by actin and calculated as a relative expression level. Each column represents the mean ± S.E.M. of 5 for control- or 11 for oxazolone-administered mice.
3.5. FAK expression is localized in colonic epithelium but not lamina propria We examined the localization of FAK in colonic tissues of control and oxazolone-administered mice by a immunohistochemical technique. In both tissues, we observed FAK immunoreactivity in
Fig. 3. Correlation of clinical scores with FAK expression level. Entire colons were dissected from 16 oxazolone-administered mice and the degree of inflammation was assessed. Also, the relative expression level of FAK in the epithelial cell lysate was measured by Western blot.
Fig. 1. Immuno-reactive proteins with YK34 in epithelial cell lysates. The epithelial cell lysate that was prepared from the control (left panel) or oxazolone-administered (right panel) mouse was separated by 2D-GE and transferred onto a PVDF membrane. The membrane was incubated with YK34 and the bound antibody was detected by ECL. Spot intensity that changed between the two panels is indicated by arrowhead.
the epithelium, but not lamina propria (Fig. 4). Remarkably, FAK was not detected in the inflammatory infiltrate in the oxazoloneadministered tissue. Furthermore, we found significantly strong immunoreactivity in scattered epithelial cells located in destroyed epithelium (Fig. 4E, arrowheads).
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4. Discussion In the present study, we found that protein expression of FAK in the colonic epithelial cell increased with the administration of oxazolone to mice, which was known to induce an acute and fulminated colitis. Although IBD usually has a slow development and chronic course, the oxazolone-induced colitis was characterized as a model for human ulcerative colitis, based on a rapidly developing Th2-mediated colitis formation [16,20,26]. The etiology of IBD is largely unknown, however, it has been shown that PTK activity in inflamed colonic mucosa was significantly higher than normal mucosa [27]. Furthermore, it has been reported that colonic gene and/or protein expression levels of PTKs, such as c-MET [28], EGFR [29], ErbB [30], Tie-2 [31], IGF1R [32], and RON [33] were changed during inflammation. To find PTK(s) involved in colitis formation, we used the monoclonal antibody,
YK34, which was raised against a highly conserved domain among a variety of PTKs [15]. We found that more than 10 immuno-reactive spots were detected. Although the intensity of most of the spots did not change regardless of the administration of oxazolone, we identified one spot in which intensity increased in the induction of colitis in a reproducible manner (Fig. 1). Based on the information of the spots in 2D-GE and Western blot, we identified FAK as a candidate that may have some relevance in the induction of colitis. FAK is well known as one of the non-receptor tyrosine kinases and has an important role in signal transduction pathways of integrin-mediated cell adhesion. FAK interacts with not only integrins but also various adaptor proteins and growth factor receptors, such as EGF, PDGF and HGF receptors at focal adhesion. The kinase activity of FAK can be regulated by these growth factors and its activation causes the interaction and/or tyrosine phosphorylation of
Fig. 4. Immunohistochemical examination of the colonic sections. The colon tissues from control mice (A and C) and oxazolone-administered mice (B, D and E) were analyzed as described in Section 2 with 40 magnification (A and B), 100 (C and D) and 200 (E).
Y. Ma et al. / FEBS Letters 584 (2010) 3949–3954
target proteins, resulting in cell detachment and migration through reorganization of focal adhesion [34,35]. Our results clearly showed the increase of FAK expression in the colonic epithelial cells of the oxazolone-administered mice. The expression of FAK is regulated by several growth factors and cytokines. The mechanism of this increase is still unknown. However, several possible mechanisms could be supposed. One mechanism is that cytokines or growth factors secreted from immune cells may activate the transcription of the FAK gene in epithelial cells. In the oxazolone colitis mouse model, the production of Th2-cytokines and growth factors, such as IL-4, IL-13 and TGFb, is highly enhanced. It is known that these cytokines enhance the transcription of various genes via the JAK/ STAT pathway. In addition, Walsh et al. reported that TGFb enhanced FAK expression via Smad and p38 mitogen-activated protein kinase (MAPK) dependent pathways in human gastric and colonic ulcers [25]. Therefore, FAK expression in epithelial cells of oxazolone-administered mice may be enhanced by the same pathway. Another mechanism is that the inert immune response of colonic epithelial cells may cause the enhancement of FAK protein expression. Colonic epithelial cells have important molecules for the inert immune system, Toll-like receptors and NOD molecules. The activation of these proteins by pathogenic bacterial components causes activation of NF-jB pathways. Golubovskaya et al. reported that the 50 -flanking region of FAK contains several potential transcription factor binding sites, and transcriptional activity of FAK was regulated by NF-jB [36]. Furthermore, both MAPK and the NF-jB pathways were also strongly activated in IBD [37]. These arguments are consistent with our results. A third mechanism is related to the correlation between FAK expression level and severity of colitis. It has been suggested that levels of FAK protein expression and/or activation have been correlated to intestinal epithelial motility [38,39]. In gastric ulcers, FAK plays an important role in wound healing [23,24]. TNF-a, which is well known as a proinflammatory cytokine and plays a key role in the pathogenesis of IBD, has been shown to activate FAK by binding to the receptors, TNFR1 and TNFR2 [40,41], and to induce an increase in FAK transcription [36]. Because we found that FAK immunoreactivity in inflamed colonic tissues was detected only in the epithelium, our results implied the functions of FAK for ulcer formation or wound healing in an inflamed colon and for pathogenesis in ulcerative colitis. Lastly, the increase in FAK expression could be a cause, and at the same time, be a consequence of colitis. It still remains to be clarified when and/or in which step FAK functions in this colitis model. Also, an alteration of FAK expression should be examined in other colitis models, which have a slower onset and chronic time course, such as a drug-induced model, IL-2 or IL-10 knock out mice, IL-7 transgenic mice [42], in order to uncover the relevance of FAK in colitis formation and in the pathogenesis of IBD. Acknowledgements This work was supported in part by National Project ‘‘Knowledge Cluster Initiative” (2nd stage, ‘‘Sapporo Biocluster Bio-S”) (T.T.) and Grant-in-Aid for Young Scientists (B) (S.S.) granted by the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Smoking Research Foundations (T.T.), and the fund for Asahikawa Medical University Creative Research in the Field of Life Science (S.S.). References [1] Bouma, G. and Strober, W. (2003) The immunological and genetic basis of inflammatory bowel disease. Nat. Rev. Immunol. 3, 521–533.
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Oxazolone-induced over-expression of focal adhesion kinase in colonic epithelial cells of colitis mouse model Yanju Ma a, Shingo Semba a, Atsuo Maemoto b, Masayuki Takeuchi a, Isamu Kameshita c, Atsuhiko Ishida d, Sanae Kato a, Tsuyoshi Katoh a, Yunpeng Liu e, Takanobu Taniguchi a,* a
Department of Biochemistry, Asahikawa Medical University, Asahikawa 078-8510, Japan Department of Internal Medicine, Asahikawa Medical University, Asahikawa 078-8510, Japan Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan d Laboratory of Molecular Brain Science, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan e Department of Medical Oncology, The First Hospital of China Medical University, Shenyang 110001, China b c
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Article history: Received 4 June 2010 Revised 9 July 2010 Accepted 27 July 2010 Available online 1 August 2010 Edited by Lukas Huber Keywords: Inflammatory bowel disease Ulcerative colitis Crohn’s disease Protein tyrosine kinase Focal adhesion kinase
a b s t r a c t We examined the change of protein tyrosine kinases (PTKs) expression levels in colonic epithelial cells isolated from mice in which colitis was induced by oxazolone administration, using the monoclonal antibody YK34, which cross-reacts with a wide variety of PTKs. We identified focal adhesion kinase (FAK) and found the expression level increased due to the induction of colitis. Furthermore, we found that there was a positive correlation between FAK expression and the severity of colitis. Also, FAK expression localized in the colonic epithelium but not in the lamina propria, implying FAK functions in epithelial cells during colitis formation and/or wound repairing. Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Ulcerative colitis and Crohn’s disease are known as inflammatory bowel diseases (IBD), which are characterized by the destruction of the gastrointestinal tract integrity, the excessive production of cytokines, and subsequent chronic intestinal inflammation. The dysregulation of the mucosal immune system was thought to play an important role during the initiation of inflammation [1]. In Crohn’s disease, T helper cell type 17 (Th17) cytokine interleukin-17 (IL-17), and Th1 cytokines interferon-c and TNF-a increased [2,3]. In ulcerative colitis, IL-17 and Th2 cytokines usually increased [2–4]. Therefore, the over-activation of immune cells in intestinal mucosa could cause chronic intestinal inflammation. Although IBD is thought to be related to genetic alterations [5] and environmental factors [6,7], their etiology and pathologic mechanisms remain largely unclear. In the mucosal immune system, the intestinal epithelium has important functions: as a physical barrier separating luminal bacteria and immune cells, and as an immunological barrier Abbreviations: IBD, inflammatory bowel disease; PTK, protein tyrosine kinase; FAK, focal adhesion kinase * Corresponding author. Fax: +81 166 68 2349. E-mail address: [email protected] (T. Taniguchi).
producing antimicrobial peptides and cytokines. The goblet cells secrete a layer of mucus that protect the intestinal epithelial cells from bacteria [8]. Disruption of epithelial integrity by dysregulation of the NF-jB pathway causes spontaneous chronic inflammation because of translocation of bacteria into mucosa [9]. Antimicrobial peptide expression by epithelial cells controls the microflora homeostasis [10,11]. Thus, the reduction of its expression is thought to be one of the causes of IBD [12]. It is also known that Toll-like receptors (TLRs), which are activated by intestinal bacteria and play a major role in the pathogenesis of IBD, are upor down-regulated in intestinal epithelial cells [13,14]. It has been believed that protein tyrosine kinases (PTKs) have critical roles for many cellular functions including immune response, ulcer formation and wound healing in the intestinal epithelium. In a previous study, Sugiyama et al. produced a monoclonal antibody, YK34, which cross-reacted with a wide variety of PTKs [15]. They used antigenic peptides corresponding to a highly conserved region among various PTKs to produce YK34. In addition, they detected the change of the PTK expression during differentiation of a leukemia HL60 cell by Western blot using YK34. We hypothesized that the PTKs expression changed with the dysregulation of intestinal epithelial functions caused by inflammation. We therefore induced chemical colitis in mice colons by intrarectal
0014-5793/$36.00 Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2010.07.054
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administration of oxazolone and examined the change of immunoreactive proteins with YK34 in colonic epithelial cell lysate by twodimensional gel electrophoresis (2D-GE) and Western blot. A database search using a determined pI value and the molecular weight of immuno-reactive spots identified focal adhesion kinase (FAK) as one of the PTKs in which the expression level was correlated with the severity of colitis. We also found that FAK was localized in epithelial cells regardless of the colitis induction. Our results suggest that FAK is involved in colitis formation and/or colonic epithelial inflammation. 2. Materials and methods 2.1. Materials 4-Ethoxymethylene-2-phenyl-2-oxazolin-5-one (oxazolone) was purchased from Sigma Chemical (St. Louis, MO, USA). Oxazolone was dissolved in 50% ethanol to obtain a 1% (w/v) solution. YK34 was raised against a highly conserved region (subdomain VIB) of PTKs [15]. Anti-FAK and -Fer antibodies were purchased from Abcam (Cambridge, UK). Anti-Syk and -b-actin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase-conjugated goat anti-rabbit antibody was purchased from Pierce (Rockford, IL, USA). 2.2. Induction of oxazolone colitis We induced colitis in Male BALB/c mice (weighing 20–25 g, which were bred in the Animal Care Facility of Asahikawa Medical University) by intrarectal administration of 100 ll of oxazolone solution via catheter delivered 4 cm into the colon [16]. Twentyfour hours after the administration, all the colonic tissues were dissected and used for further experiments. The degree of inflammation was assessed by macroscopic observation as described previously [17] using the following criteria: 0 – no inflammation; 1 – swelling or redness; 2 – swelling and redness; 3 – one or two ulcers; 4 – more than two ulcers or one large ulcer; 5 – mild necrosis; 6 – severe necrosis. All animal procedures were approved by the Animal Care Committees of Asahikawa Medical University.
7 M urea, 2 M thiourea, 5 mM Tris–HCl, 0.5% Nonidet P-40 and 5 mM DTT. Isoelectric focusing (IEF) was carried out using agarose IEF gels (AgarGEL, pH 3–8, ATTO, Tokyo, Japan) in the first dimension and 7.5% polyacrylamide slab gels in the second dimension according the manufacturer’s guidelines. 2.5. Western blot Proteins that were separated by gel electrophoresis were transferred to a Immobilon-P PVDF membrane (Millipore, Billerica, MA, USA). The membrane was blocked with Tris-buffered saline containing 0.05% Tween 20 (TBST) and 2% ECL Advance Blocking Reagent (GE Healthcare UK Ltd., Buckinghamshire, England). After incubation with the appropriate primary antibody, bound antibody was detected by using peroxidase-conjugated secondary antibody and ECL Advance Western Blotting Detection Kit (GE Healthcare). 2.6. Immunohistochemistry Colonic tissues dissected from mice were fixed with 20% buffered formalin and embedded in paraffin, and cut into tissue sections. After deparaffinization, each section was incubated with 1:100 dilution of anti-FAK antibody for 1 h at room temperature and then washed three times with PBS. Bound primary antibody was detected by using anti-rabbit antibody and diaminobenzidine staining. 2.7. Analysis of data The level of FAK protein expression was measured by densitometric measurement of the immuno-reactive signal with antiFAK antibody of the Western blots using ImageJ software. FAK expression levels were normalized based on b-actin expression levels in same blots. Results are expressed as the means ± S.E.M. of n experiments. Statistical significance was evaluated using Student’s t-test. P < 0.05 was considered statistically significant. The correlation between the clinical score and FAK expression level was evaluated by using Pearson product-moment correlation coefficient.
2.3. Preparation of colonic epithelial cells and its lysate 3. Results We prepared colonic epithelial cells as described [18], in which the purity of the colonic epithelial cell preparation has been shown to be approximately 90% [19]. Briefly, the dissected colon was opened by cutting longitudinally and gently rinsed with ice-cold phosphate-buffered saline (PBS). Then the colon was cut into pieces of about 5 mm 5 mm and transferred into a 2.0-ml microtube containing 1.5 ml of PBS. The tube was vortexed for 5 min at room temperature. After removal of the supernatant, 1 ml of PBS containing 20 mM EDTA was added into the tube. After vortexing for 10 min at room temperature, the cell suspension was collected by centrifugation. This step was repeated 6–8 times. Next, the cells were suspended in 33% Percoll and centrifuged for 20 min at 400g. After centrifugation, the cells on the top of the 33% Percoll were collected and used as epithelial cells. Then, the cells were suspended in five volumes of lysis buffer containing 0.5 M NaCl, 0.2 mM EDTA, 1.5 mM MgCl2, 1 mM DTT, 1 mM PMSF and 20 mM Tris–HCl, pH 7.5 and lysed by passing through a syringe equipped 22-gauge needle repeatedly. After centrifugation at 12 000g for 20 min at 4 °C, the lysate was used for the following experiments.
3.1. Induction of colitis by the administration of oxazolone We employed the oxazolone-induced colitis model that was accepted as a T helper-2 type model [20,21] similar to human ulcerative colitis. Since we found that the mortality of the oxazoloneadministered mice by the end of day 2 was approximately 50%, we examined the severity of colitis at 24 h posttreatment. At this time point, some oxazolone-administered mice showed soft stool, rectal bleeding and fecal staining around the anus. Macroscopic examination revealed that inflammation had occurred in the distal colon. However, the severity varied widely; about 30% of the oxazolone-administered mice showed no colitis, redness or swelling of the bowel wall (clinical score 0 and 1), whereas 40% exhibited mild to moderate inflammation (clinical score 2–4) and the other 30% displayed large ulcers accompanied with necrosis (clinical score 5 and 6). 3.2. Detection of immuno-reactive proteins with YK34 antibody in colonic epithelial cell lysate
2.4. Two-dimensional gel electrophoresis Cell lysates were desalted by trichloroacetic acid precipitation and the precipitates were dissolved in a sample buffer containing
In order to investigate the change of the PTK expression level, we carried out 2D-GE and Western blot analysis using the monoclonal antibody YK34. Twenty-four hours after the administration
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of oxazolone, the epithelial cell lysate was prepared from each colonic tissue and analyzed by Western blot. As shown in Fig. 1, more than 10 immuno-reactive spots were detected. Among them, we found that the intensity of the spot, of which the molecular mass was 120 kDa and pI value was 6.3, was significantly and reproducibly increased by the administration of oxazolone (Fig. 1, arrowheads). On the basis of these values, we searched the UniProtKB/ Swiss-Prot database using the TagIdent program [22] with the following: tyrosine-protein kinases of Mus musculus with a molecular mass 120 000 ± 12 000 Da and pI value between 6.0 and 6.5. Several PTKs including FAK 1 were suggested by the search. 3.3. Oxazolone administration enhances FAK expression in epithelial cells Previous studies showed that FAK contributes to wound healing in gastric injuries [23,24] and FAK expression increased in epithelial cells near human gastric and colonic ulcers [25]. We thus examined the protein expression level of FAK in the colon epithelium. Lysates were prepared from colonic epithelial cells in mice and were subjected to Western blot using anti-FAK antibody. As shown in Fig. 2A, the expression level of FAK after oxazolone administration was significantly higher in all lysates prepared from inflamed colonic tissues compared with those from control tissues. In contrast, other PTKs such as Syk and Fer did not show any change of their expression levels. Densitometric analysis revealed that FAK expression in oxazoloneadministered mice increased to 168 ± 15.5% compared with ethanol-administered mice (P = 0.011, Fig. 2B). 3.4. FAK expression level correlates with severity of oxazolone-induced colitis Using Western blot analysis, we found the variance in the expression level of FAK in inflamed colonic epithelial cell lysates. In order to examine the correlation between the severity of colitis and FAK expression level, we assessed the degree of inflammation of each oxazolone-administered mouse colon. The estimated clinical score was plotted as a function of the relative expression level of FAK (Fig. 3). The correlation between the clinical score and FAK expression was modest (r = 0.681).
Fig. 2. Changes of the protein expression levels of FAK in epithelial cell lysates. (A) Representative results of Western blot. Lysates that were prepared from the colonic epithelial cells of control mice (lanes marked as ) or oxazolone-administered mice (lanes marked as +) were probed with anti-FAK, -Syk and -Fer antibodies in Western blot. The b-actin was also detected as a loading control. (B) Quantification of FAK expression level. Optical density of each band was measured by densitometry. The expression levels of FAK were then normalized by actin and calculated as a relative expression level. Each column represents the mean ± S.E.M. of 5 for control- or 11 for oxazolone-administered mice.
3.5. FAK expression is localized in colonic epithelium but not lamina propria We examined the localization of FAK in colonic tissues of control and oxazolone-administered mice by a immunohistochemical technique. In both tissues, we observed FAK immunoreactivity in
Fig. 3. Correlation of clinical scores with FAK expression level. Entire colons were dissected from 16 oxazolone-administered mice and the degree of inflammation was assessed. Also, the relative expression level of FAK in the epithelial cell lysate was measured by Western blot.
Fig. 1. Immuno-reactive proteins with YK34 in epithelial cell lysates. The epithelial cell lysate that was prepared from the control (left panel) or oxazolone-administered (right panel) mouse was separated by 2D-GE and transferred onto a PVDF membrane. The membrane was incubated with YK34 and the bound antibody was detected by ECL. Spot intensity that changed between the two panels is indicated by arrowhead.
the epithelium, but not lamina propria (Fig. 4). Remarkably, FAK was not detected in the inflammatory infiltrate in the oxazoloneadministered tissue. Furthermore, we found significantly strong immunoreactivity in scattered epithelial cells located in destroyed epithelium (Fig. 4E, arrowheads).
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4. Discussion In the present study, we found that protein expression of FAK in the colonic epithelial cell increased with the administration of oxazolone to mice, which was known to induce an acute and fulminated colitis. Although IBD usually has a slow development and chronic course, the oxazolone-induced colitis was characterized as a model for human ulcerative colitis, based on a rapidly developing Th2-mediated colitis formation [16,20,26]. The etiology of IBD is largely unknown, however, it has been shown that PTK activity in inflamed colonic mucosa was significantly higher than normal mucosa [27]. Furthermore, it has been reported that colonic gene and/or protein expression levels of PTKs, such as c-MET [28], EGFR [29], ErbB [30], Tie-2 [31], IGF1R [32], and RON [33] were changed during inflammation. To find PTK(s) involved in colitis formation, we used the monoclonal antibody,
YK34, which was raised against a highly conserved domain among a variety of PTKs [15]. We found that more than 10 immuno-reactive spots were detected. Although the intensity of most of the spots did not change regardless of the administration of oxazolone, we identified one spot in which intensity increased in the induction of colitis in a reproducible manner (Fig. 1). Based on the information of the spots in 2D-GE and Western blot, we identified FAK as a candidate that may have some relevance in the induction of colitis. FAK is well known as one of the non-receptor tyrosine kinases and has an important role in signal transduction pathways of integrin-mediated cell adhesion. FAK interacts with not only integrins but also various adaptor proteins and growth factor receptors, such as EGF, PDGF and HGF receptors at focal adhesion. The kinase activity of FAK can be regulated by these growth factors and its activation causes the interaction and/or tyrosine phosphorylation of
Fig. 4. Immunohistochemical examination of the colonic sections. The colon tissues from control mice (A and C) and oxazolone-administered mice (B, D and E) were analyzed as described in Section 2 with 40 magnification (A and B), 100 (C and D) and 200 (E).
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target proteins, resulting in cell detachment and migration through reorganization of focal adhesion [34,35]. Our results clearly showed the increase of FAK expression in the colonic epithelial cells of the oxazolone-administered mice. The expression of FAK is regulated by several growth factors and cytokines. The mechanism of this increase is still unknown. However, several possible mechanisms could be supposed. One mechanism is that cytokines or growth factors secreted from immune cells may activate the transcription of the FAK gene in epithelial cells. In the oxazolone colitis mouse model, the production of Th2-cytokines and growth factors, such as IL-4, IL-13 and TGFb, is highly enhanced. It is known that these cytokines enhance the transcription of various genes via the JAK/ STAT pathway. In addition, Walsh et al. reported that TGFb enhanced FAK expression via Smad and p38 mitogen-activated protein kinase (MAPK) dependent pathways in human gastric and colonic ulcers [25]. Therefore, FAK expression in epithelial cells of oxazolone-administered mice may be enhanced by the same pathway. Another mechanism is that the inert immune response of colonic epithelial cells may cause the enhancement of FAK protein expression. Colonic epithelial cells have important molecules for the inert immune system, Toll-like receptors and NOD molecules. The activation of these proteins by pathogenic bacterial components causes activation of NF-jB pathways. Golubovskaya et al. reported that the 50 -flanking region of FAK contains several potential transcription factor binding sites, and transcriptional activity of FAK was regulated by NF-jB [36]. Furthermore, both MAPK and the NF-jB pathways were also strongly activated in IBD [37]. These arguments are consistent with our results. A third mechanism is related to the correlation between FAK expression level and severity of colitis. It has been suggested that levels of FAK protein expression and/or activation have been correlated to intestinal epithelial motility [38,39]. In gastric ulcers, FAK plays an important role in wound healing [23,24]. TNF-a, which is well known as a proinflammatory cytokine and plays a key role in the pathogenesis of IBD, has been shown to activate FAK by binding to the receptors, TNFR1 and TNFR2 [40,41], and to induce an increase in FAK transcription [36]. Because we found that FAK immunoreactivity in inflamed colonic tissues was detected only in the epithelium, our results implied the functions of FAK for ulcer formation or wound healing in an inflamed colon and for pathogenesis in ulcerative colitis. Lastly, the increase in FAK expression could be a cause, and at the same time, be a consequence of colitis. It still remains to be clarified when and/or in which step FAK functions in this colitis model. Also, an alteration of FAK expression should be examined in other colitis models, which have a slower onset and chronic time course, such as a drug-induced model, IL-2 or IL-10 knock out mice, IL-7 transgenic mice [42], in order to uncover the relevance of FAK in colitis formation and in the pathogenesis of IBD. Acknowledgements This work was supported in part by National Project ‘‘Knowledge Cluster Initiative” (2nd stage, ‘‘Sapporo Biocluster Bio-S”) (T.T.) and Grant-in-Aid for Young Scientists (B) (S.S.) granted by the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Smoking Research Foundations (T.T.), and the fund for Asahikawa Medical University Creative Research in the Field of Life Science (S.S.). References [1] Bouma, G. and Strober, W. (2003) The immunological and genetic basis of inflammatory bowel disease. Nat. Rev. Immunol. 3, 521–533.
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