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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Department of Internal Medicine, Section of Gastroenterology (C.B.F., A.B., A.F., J.Z.F., Y.T., M.S., L.J.Z., P.A.E., A.K.) and Departments of Pharmacology (A.B., A.K.) and Physiology (A.K.), Rush University Medical Center, Chicago, Illinois
Received for publication
August 28, 2006
Accepted
January 11, 2007.
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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). The aim of this study was to investigate the role of oxidant stress-mediated transactivation of the epidermal growth factor receptor (EGFR) in intestinal hyperpermeability. This study used the Caco-2 human colonic epithelial cell in vitro model of intestinal epithelium. Cells were grown on inserts for permeability and signaling studies and glass coverslips for microscopy studies. show that oxidant-induced intestinal hyperpermeability can be blocked by specific inhibitors of the EGFR, tumor necrosis factor convertase (TACE) metalloprotease, transforming growth factor (TGF)-, and mitogen-activated protein kinases, especially extracellular signal-regulated kinase 1/2. We also show that oxidant initiates these signaling events, in part by causing translocation of TACE to cell-cell contact zones. In this study, our data identify a novel mechanism for oxidant-induced intestinal hyperpermeability relevant to IBD. We propose a new intestinal permeability model in which oxidant transactivates EGFR signaling by activation of TACE and cleavage of precursor TGF-. These data could have a significant effect on our view of IBD pathogenesis and provide new therapeutic targets for IBD treatment.
; Farhadi et al., 2003; Clayburgh et al., 2004) This disorder profoundly increases patient morbidity and decreases quality of life and increases patient susceptibility to colorectal cancer, although its etiology remains unknown. Current models of disease pathogenesis identify as the two principal determinants of IBD pathogenesis: increased permeability of the epithelial barrier of the intestinal mucosa, which results in penetration of luminal bacterial products into the mucosa; and an abnormal immune response to these products (Farhadi et al., 2003; Clayburgh et al., 2004). Regarding intestinal hyperpermeability in IBD, two factors are thought to play critical roles: oxidant-induced stress and proinflammatory cytokines, especially TNF- and interferon-
(Bruewer et al., 2003; Podolsky, 2002). The principal permeability pathway in IBD is the paracellular route, which is ultimately regulated by tight junctions (TJs) between the intestinal epithelial cells (IECs) that constitute the intestinal barrier (Clayburgh et al., 2004). Clearly, a better understanding of the mechanisms underlying oxidant-induced barrier disruption and intestinal hyperpermeability seems to be a key to understanding the pathogenesis of IBD. Therefore, we studied these mechanisms in the hope that it would suggest targets for novel interventions that interrupt pathogenic inflammatory cascades in IBD.
The study being reported herein is part of our long-term goal of identifying mechanisms of oxidant-induced intestinal hyperpermeability using in vitro, animal, and clinical models of IBD (Keshavarzian et al., 1990; Banan et al., 2000a,b). Using in vitro models of human intestinal epithelium, this and other laboratories have identified key roles for specific intracellular signaling pathways mediating disruption of TJ permeability by oxidative stress. These pathways involve signaling via the epidermal growth factor receptor (EGFR) (Banan et al., 2000a,b, 2001a,b), signaling via NF-kB activation (Banan et al., 2004), and signaling via specific protein kinase C (PKC) isoforms, which are either protective or injurious, depending on the PKC isoform (Banan et al., 2001b, 2005). Previous studies investigating a possible role for the EGFR in oxidant-induced intestinal hyperpermeability have determined that preincubation with epidermal growth factor (EGF) or transforming growth factor (TGF)- before exposure to oxidant (H2O2) prevents oxidant-induced intestinal hyperpermeability (Banan et al., 2001b, 2002; Basuroy et al., 2006).
Recently, a new pathway for oxidant (H2O2) signaling through EGFR activation was identified. The pathway is "metalloprotease-dependent transactivation of EGFR signaling" (Prenzel et al., 1999; Fischer et al., 2004). In this model (see Fig. 15), metalloproteases, especially from the ADAM family of membrane-bound metalloproteases, become activated by a variety of signals and cleave EGFR precursor ligands attached to the cell surface. These solubilized ligands then bind to the EGFR and initiate EGFR-mediated signaling pathways (Schafer et al., 2004). Several studies have shown that oxidants (e.g., H2O2) are one of several stimuli capable of triggering signaling via ADAM-dependent transactivation of the EGFR (Schafer et al., 2004). The ADAM involved in greater than 80% of all cases is the tumor necrosis factor- convertase (TACE; ADAM-17) (Black et al., 1997; Blobel, 2005). In the intestine, TACE levels are increased in the inflamed mucosa of patients with IBD, and TACE inhibitors ameliorate colitis in animal models of IBD (Colon et al., 2001; Brynskov et al., 2002). We hypothesized that oxidant-induced metalloprotease-EGFR signaling could play a role in oxidant-induced increases in the intestinal permeability characteristic of IBD.
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in response to oxidant stress. We also identify downstream EGFR-mediated mitogen-activated protein kinase (MAPK) signaling via ERK1/2 MAPKs to be necessary for oxidant-induced intestinal hyperpermeability. These data are the first demonstration of this EGFR transactivation mechanism regulating intestinal epithelial permeability. In addition, oxidant stress is known to exist in IBD epithelium, and TACE is required for TNF- production so important in IBD pathogenesis. Therefore, stimulation of TACE activity by oxidant stress could clearly fuel IBD pathogenesis by stimulation of both TNF- production and now we show through EGFR-mediated increased permeability resulting from TACE cleavage of TGF-. This study could therefore have significant implications toward a new understanding of IBD pathogenesis in addition to providing new avenues for therapy of IBD. |
Materials and Methods |
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(R&D Systems), and the CRM197 HB-EGF blocker (diphtheria toxin) (Sigma). We also used Ab to ZO-1 (Invitrogen, Carlsbad, CA) and a rabbit polyclonal anti-TACE Ab (Sigma). EGFR (Ab-1), phosphotyrosine Ab (4G10), and EGFR-positive control were from Upstate Biotechnology. All other phospho- and total MAPK Abs and controls were from Cell Signaling Technology (Danvers, MA). These comprised phosphospecific Abs, including ERK1/2, p38, JNK1/2, and AKT and total MAPK Abs (controls for loading), including ERK1/2, p38, JNK1/2, and AKT. Cell Culture. We used confluent human colonic epithelial Caco-2 cells (number CRL2101, C2BBe1, colorectal adenocarcinoma; American Type Culture Collection, Manassas, VA) or IEC-6 (rat, nontransformed intestinal epithelial; American Type Culture Collection number CRL1592) between passages 15 and 25 grown on permeable culture inserts (Transwell; Corning, Corning, NY) for permeability and signaling studies and grown on glass coverslips for microscope-based imaging studies. Cells were cultured at 37°C/5% CO2 in a humidified incubator in Dulbecco's modified Eagle's medium (DMEM)/10% fetal bovine serum media with 5 mM penicillin-streptomycin and 0.01 mg/ml human transferrin.
Determination of Integrity of the Intestinal Permeability Barrier Using Monolayers of Caco-2 Cells. Barrier permeability was determined using Caco-2 cells grown to confluence on 6.5-mm/0.2-µm 24-well tissue culture plate inserts (Transwell; Corning) as we described previously (Banan et al., 2000a,b). Permeability of insert Caco-2 monolayers was measured as apical to basolateral flux of the fluorescent marker fluorescein-5-(and-6)-sulfonic acid trisodium salt (FSA, 478 Da) (Invitrogen) or fluorescein isothiocyanate (FITC)-dextran (4 kDa) (Sigma). This is an assay whose utility and validity have been shown and extensively used by others and us and described previously (Sanders et al., 1995; Banan et al., 2001a,b). In brief, phenol red-free, serum-free DMEM (800 µl) was added into the bottom (basolateral) chamber, and DMEM (300 µl) with FSA (400 nM) or FITC-dextran (1.25 µM) was added into the top (apical) chamber. At specific times, aliquots (50 µl) were transferred to opaque-bottom, 96-well fluorescence plates. Fluorescent signals were quantitated using a fluorescence plate reader and reported as "FSA or FITC-dextran clearance" (nanomoles per centimeter squared per hour). Excitation/emission spectra for FSA/FITC-dextran were 485/530 nm. Controls and a standard curve were done with each run. Inhibitors and blocking Abs were preincubated with cells for 1 h before adding H2O2.
Western Blotting Analysis. Western blotting was performed using the Bio-Rad Mini-Protean 3 system (Bio-Rad, Hercules, CA) as described previously (Forsyth et al., 2002). Cells were lysed with RIPA buffer containing protease and phosphatase inhibitor cocktails (Sigma) and 1 mg/ml phenylmethanesulfonyl fluoride with 1% Non-idet P-40. Lysates were assessed for total protein (Bio-Rad), and 20 µg was loaded per lane of 4/10% 1.0 mM Laemmli SDS-polyacrylamide gel electrophoresis gels. Protein was transferred to nitrocellulose and blocked with 5% bovine serum albumin for 1 h at 4°C and then incubated at 4°C overnight with primary Ab. Blots were then washed, incubated with horseradish peroxidase-conjugated 2°C Ab for 2 h at 4°C, washed, developed with enhanced chemiluminescence (GE Healthcare, Piscataway, NJ), exposed to film (Fuji, Tokyo, Japan), and scanned for densitometry analysis with NIH Image J software (Banan et al., 2005).
Isolation of RNA and RT-PCR. Isolation of total RNA was done using the RNEasy kit (QIAGEN, Valencia, CA). RNA was quantitated in a spectrometer. Reverse transcription of RNA to cDNA was carried out using the High-Capacity cDNA RT kit (Applied Biosystems, Foster, CA). Real-time quantitative PCR was then performed using the resulting cDNA with the Sybr Green PCR Master Mix (Applied Biosystems) and 35 cycles of 95 to 60°C and 72°C (each 1 min) for PCR amplification. The reactions were performed in 96-well plates using an Applied Biosystems 7900HT Prism apparatus. Primers for RT-PCR used in the present study were: 
-actin (157 bp), forward, 5'-GCCAGGTCCAGACGCAGG-3' and reverse, 5'-TGCTATCCAGGCTGTGCTA-3'; and TACE (154 bp), forward, 5'-CTGTGGTGCAAAAGCAGAAA-3' and reverse, 5'-TGCCAAATGCCTCATATTCA-3'. Primers were designed with Oligoperfect software (Invitrogen) and purchased from Invitrogen. TACE data were normalized first to actin expression in the same sample and then normalized to the value of TACE expression in the "0 time" start cells to arrive at the -fold expression value.
Short Inhibitory RNA Inhibition Studies. Smartpool short inhibitory RNA (SiRNA) specific for human ADAM-17 (TACE), and control SiRNA were purchased from Dharmacon, Inc. (Lafayette, CO). Caco-2 cells grown to 60% confluence in six-well plates were transfected with 60 pmol/ml ADAM-17 SiRNA or control SiRNA using Lipofectamine (Invitrogen) and Opti-MEM reduced serum media (Invitrogen) according to manufacturer's instructions. At 7 days post-treatment, cells were assessed for expression of TACE protein using Western blotting. Cells with greater than 80% knockdown of TACE expression were then treated with H2O2 and assessed for ERK1/2 signaling and TACE expression by Western blot.
Microscopy. Microscopic images were obtained of Caco-2 cells grown to confluence plus 7 days on glass coverslips using an Axiovert 100 microscope and Axiovision software (Carl Zeiss Inc., Thornwood, NY). Two-dimensional images (see Figs. 10, 11, 12) were obtained using an oil immersion 100x objective, whereas images for Figs. 13 and 14 were obtained using an oil immersion 40x objective. Figures 13 and 14 show images obtained by deconvolution of z-stack images (15 at 1 µm each). Three-dimensional reconstruction of deconvoluted z-stacks was performed using the Zeiss Axiovision software (Carl Zeiss Inc.). Immunofluorescent microscopic analysis of Caco-2 monolayers has been described previously by us in greater detail (Banan et al., 2001a,b, 2004, 2005).
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0.05 being considered significant. Data were analyzed using SPSS software for Windows (SPSS, Chicago, IL). |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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) in protection against IEC permeability caused by oxidative stress (Banan et al., 2000a,b, 2001a,b; Basuroy et al., 2006). In addition, recent discoveries have shown that metalloprotease-EGFR signaling mediates cellular response to H2O2 in other cell types (Fischer et al., 2004). Therefore, we sought to determine whether metalloprotease-EGFR signaling could be playing a role in increased permeability caused by oxidative stress in IBD. We used the in vitro Caco-2 insert monolayer model that has been well established by this and other laboratories as a reliable in vitro model of intestinal permeability (Banan et al., 2000a,b; Basuroy et al., 2006). Permeability of monolayers was monitored by addition of the fluorescent dye fluorescein sulfonic acid (FSA; molecular mass, 478 Da) added at a concentration of 400 nM to the top chamber and fluorescence of the bottom chamber media (phenol red-free and serum-free DMEM) was assessed at 18 h, and the data were displayed in Fig. 1 as "FSA clearance" (change in FSA at 490/530 nM in the bottom well) in nanomoles per centimeter squared per hour (Sanders et al., 1995; Banan et al., 2005). All permeability data are means of triplicate wells from a representative experiment of more than three such experiments. Using H2O2 as a model stimulus for oxidative stress, we subjected cells to 500 µ MH2O2. This dose of H2O2 is consistent with the 200 to 500 µM range of H2O2 used by ourselves in previous studies and other investigators as a model stimulus of oxidative stress (Fischer et al., 2004; Song et al., 2006). We have documented in previous studies that this dose does not significantly affect cell viability (Banan et al., 2000a,b). This treatment caused a dramatic increase (
5-fold/h) in monolayer permeability without affecting cell viability (Fig. 1) and confirmed prior studies that oxidant stress increases Caco-2 permeability (Banan et al., 2000a,b). The development of hyperpermeability was inhibited (–66%) by the EGFR-specific tyrosine kinase inhibitors AG1478 (500 nM) (Fig. 1a, H2O2 + EGFRi) and GW2901 (data not shown). It was inhibited to a somewhat greater extent (>70%) by the broad MMP-TACE inhibitor GM6001 (20 µM) (Fig. 1a, MMPi) and the TACE-specific inhibitor TAPI-2 (10 µM) (Fig. 1a, TACEi). The inhibition was significant (p < 0.05) compared with the H2O2 + DMSO control. These two inhibitors are known to markedly inhibit ADAM-17 (TACE) at the concentrations used (Mohler et al., 1994; Santiskulvong and Rozengurt, 2003). These data strongly support a TACE metalloprotease-EGFR transactivation mechanism, one requiring TACE cleavage of EGFR proligands such as EGF, TGF-, or HB-EGF. Also shown in Fig. 1a, AG1478 (control + EGFRi) as well as GM6001 (control + MMPi) and TAPI-2 (control + TACEi) also caused small but significant (p < 0.05) increase in permeability relative to control wells treated only with media + vehicle (control + DMSO). These data support a role for TACE-EGFR signaling in regulation of baseline intestinal epithelial permeability as well. Taken together, these data strongly support a role for TACE-EGFR signaling in regulation of both the baseline permeability function as well as in response to the IBD relevant oxidative stress model stimulus H2O2.
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In light of these data, we next sought to determine whether the EGFR and TACE inhibitors could protect against oxidant-induced changes in the key tight junction protein ZO-1. We and others have previously shown that another Caco-2 model system can be used to investigate mechanisms of H2O2-induced changes in IEC permeability (Banan et al., 2005; Basuroy et al., 2006). Cells grown to confluence on glass coverslips can be treated under the same experimental conditions, fixed, and stained for the TJ protein ZO-1 (Fig. 1b, i–iv). Increased displacement ("zig-zag" pattern) of ZO-1 in this system correlates well with oxidant-induced increased monolayer permeability (Banan et al., 2005; Basuroy et al., 2006). Immunofluorescence microscopy clearly shows that the dramatic displacement of ZO-1 caused by H2O2 (Fig. 1b, ii) is prevented by pretreatment of cells with the EGFR kinase-specific inhibitor AG1478 (Fig. 1b, iii) or the TACE-specific inhibitor TAPI-2 (Fig. 1b, iv). These data further support a requirement for EGFR and TACE-mediated signaling in oxidant-induced increased barrier permeability shown in Fig. 1a.
These above data support a requirement for EGFR signaling in oxidant-induced intestinal epithelial permeability. Therefore, we next sought to determine what key downstream EGFR signaling pathways might be mediating the increase in permeability.
Key signaling pathways activated by EGFR signaling in other cell types include MAPK and AKT signaling (Fischer et al., 2004; Schafer et al., 2004). Thus, we determined the effect on H2O2-induced hyperpermeability of inhibitors of three principal MAPK pathways: ERK1/2, JNK1/2, and p38 as well as the effect of inhibition of phosphatidylinositide 3'-OH kinase activation of AKT (Fig. 1c). Of the inhibitors tested, Fig. 1c shows only the ERK1/2 inhibitors PD98059 (20 µM) and U0126 (20 µM) (data not shown) inhibited oxidant-induced permeability in a manner as great as the EGFR or MMP inhibitors in Fig. 1a. These data suggest that ERK1/2 activation is one major downstream signaling mechanism for EGFR-mediated hyperpermeability in response to H2O2 stimulation. Finally, we sought to validate the FSA permeability data with another marker of IEC permeability. We used the well validated 4-kDa FITC-dextran marker (1.25 µM) added to the apical surface of Transwells and assessed movement to the bottom chamber over 18 h as for FSA above (Fig. 1d). As for FSA, oxidant-induced permeability to 4-kDa FITC-dextran was substantially increased over 18 h, and this increase could be virtually eliminated by pretreatment with the EGFR, metalloprotease, and ERK inhibitors.
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Besides EGFR phosphorylation, the parameter most often used to assess EGFR activation/signaling is phosphorylation (activation) of the MAPKs ERK1 and ERK 2 (Prenzel et al., 1999; Schafer et al., 2004). Figure 2b shows that both EGFR and TACE inhibitors reduce ERK1/2 activation to below control levels. As expected, the mitogen-activated protein kinase kinase-1 inhibitor PD98059 also eliminates ERK1/2 activation. Taken together, the phosphoprotein signaling data from Fig. 2, a and b, support a metalloprotease-dependent transactivation model in which oxidant stress-induced EGFR phosphorylation is upstream of ERK1/2 activation and that the activity of TACE or another ADAM metalloprotease (inhibited by TAPI-2) is required for oxidant-induced EGFR phosphorylation. We also wished to establish that H2O2-induced signaling via EGFR-metalloprotease activation could be demonstrated in other IEC cell lines and not only in Caco-2 cells. To do this, we used IEC-6 cells, a nontransformed rat cell line widely used in intestinal epithelial models (Fig. 3). Confluent cells were treated with H2O2 ± an EGFR inhibitor (AG1478) or TACE inhibitor (TAPI-2) and assessed for ERK1/2 activation with phosphospecific Abs as in Fig. 2b. As with the Caco-2 cells, oxidant induced a greater than 5-fold increase in ERK1/2 phosphorylation that was inhibited by both the EGFR inhibitor (75%) and the TACE inhibitor (82%). These data further support the EGFR-metalloprotease model of H2O2-induced signaling in IECs and that it is also true for other intestinal epithelial cells.
SiRNA Studies Identify TACE as the Principal Metalloprotease Mediating H2O2-Induced EGFR Signaling in Caco-2. Because chemical inhibition of metalloproteases is not gene specific, we next sought to establish whether TACE or another metalloprotease was mediating H2O2-induced EGFR signaling in IECs using gene-specific knockdown of TACE with SiRNA (Fig. 4). We used Smartpool SiRNA from Dharmacon specific for human ADAM-17 (TACE) and used by another group to successfully knockdown TACE (Fischer et al., 2005) and transfected the SiRNA (60 pmol/ml) under optimized conditions into Caco-2 cells grown to 60% confluence. After 7 days, cells were treated with H2O2, and the response to oxidant was assessed by ERK1/2 phosphorylation. Blots were also stripped and reprobed with Ab to TACE to assess expression of TACE 110- and 90-kDa forms. The data shown in Fig. 4 reveal that in cells in which TACE protein expression was knocked down by 84% (lane 3, TACE SiRNA) compared with the control SiRNA-treated cells (lane 4), the H2O2-induced ERK1/2 activation was also reduced by 85% (p < 0.05) compared with the control SiRNA-treated cells. Our previous data above showed that H2O2-induced ERK1/2 activation in Caco-2 is EGFR- and metalloprotease-dependent and that ERK1/2 activation is required for H2O2-induced hyperpermeability. Therefore, although these data do not rule out participation of other metalloproteases, they do demonstrate in a gene-specific manner that TACE is the principal ADAM metalloprotease mediating H2O2-induced EGFR-ERK1/2 signaling and therefore increased permeability by this mechanism in Caco-2 cells.
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TACE Protein Is Abundantly Expressed by Caco-2 Cells, and EGFR and TACE Inhibitors Do Not Act by Inhibiting TACE Protein Expression. Because the above data point to a role for TACE in H2O2-induced signaling and permeability, we wished to determine whether sufficient quantities of TACE were expressed by Caco-2 cells and how EGFR, TACE, and ERK1/2 inhibitors affected TACE protein expression. TACE protein was assessed using Western blotting of cell lysates with polyclonal antibody to the TACE cytoplasmic domain (Fig. 5). Studies of other cell types including Caco-2 cells have shown that production of TACE is relatively constant and that TACE exists in the cell as an intracellular, 110-kDa, inactive zymogen, "pro-TACE" form and an activated 90-kDa form predominantly found on the cell surface or near the cell surface in intracellular vesicles (Schlondorff et al., 2000; Soond et al., 2005). The 110-kDa pro-TACE seems to be associated with the cytoskeleton in a perinuclear location (Soond et al., 2005). The data from Caco-2 cells in Fig. 3 are in lanes 2 to 6. Lane 1 "+Con" contains a TACE-positive control cell lysate (Chemicon). The 15-min time point chosen was the same as that for the EGFR and ERK1/2 signaling data for comparison. No significant differences in TACE protein expression from these data were seen with the EGFR and TACE inhibitors in 18-h lysates (data not shown). Clearly, Caco-2 cells even at rest express abundant TACE in both the pro-110-kDa as well as the activated 90-kDa forms (lane 2). In addition, treatment with H2O2 results in only a slight increase (but not statistically significant) in both the 110- and 90-kDa forms of TACE (lane 3). However, in the presence of H2O2, treatment with the EGFR inhibitor (lane 4), TACE inhibitor (lane 5), or the ERK1/2 inhibitor (lane 6) did not significantly alter the amount of pro- or activated TACE to an extent that would account for complete blocking of EGFR or ERK1/2 phosphorylation as seen in Fig. 2. In fact the amount of active 90-kDa TACE is relatively constant in lanes 2 to 6, with no statistical difference detected. The blot was stripped and reprobed with Ab to actin as a protein loading control. Taken together, these data show that strong expression of TACE protein by Caco-2 in our system is consistent with other data above pointing to a role for TACE in oxidant-induced EGFR-mediated signaling and permeability changes. However, activation of TACE-mediated EGFR signaling does not seem to be related to changes in the active form of TACE protein.
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Although no changes in TACE protein expression were seen either at 15 min or 18 h, we wished to assess whether changes in TACE gene expression (mRNA) could account for increased TACE signaling required for H2O2-induced permeability (Fig. 6). We also wished to determine whether gene expression (mRNA) of TACE was altered in cells after H2O2 treatment for 18 h ± EGFR inhibitor. In addition, cells were treated with EGF, which we have shown in previous studies to prevent H2O2-induced permeability in Caco-2 inserts (Banan et al., 2000a,b). In Fig. 6, data from quantitative real-time RT-PCR for TACE are normalized in each lane to actin expression and that ratio is expressed as a -fold expression of the start control (histogram). Treatment with H2O2 alone resulted in reduced TACE expression, although treatment with the EGFR inhibitor resulted in increased TACE expression. Interestingly, treatment with EGF also resulted in down-regulation of TACE expression. These data suggest a negative feedback mechanism that regulates TACE expression when EGFR signaling is stimulated by oxidants or EGF. Clearly, however, these data do not support increased TACE gene expression as the probable mechanism for oxidant-induced hyperpermeability through EGFR-TACE signaling.
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as the EGFR Ligand Mediating Increased Permeability in Response to Oxidant. A key element in metalloprotease-EGFR transactivation is the EGFR proligand cleaved on the surface of the cell by TACE. To identify this ligand, we assessed the effects of blocking proteins to the EGFR and EGFR ligands on hyperpermeability induced by H2O2 (Fig. 7). Blocking antibodies to amphiregulin had no effect at several concentrations (data not shown). The blocking protein for HB-EGF (CRM197, 10 µg/ml, a diphtheria toxin) had a dramatic effect on resting cells (bar 3), increasing permeability to that of H2O2-treated cells, but had little effect at blocking H2O2-induced permeability (bar 7). In contrast, blocking Ab to TGF- (10 µg/ml) had little effect on resting permeability (bar 4) but blocked H2O2-induced hyperpermeability by 96% (bar 8). Cells treated with blocking Ab to the EGFR caused some leakiness in control cells and blocked H2O2-induced hyperpermeability by 80%. These data support a role for TGF- as the oxidant-stimulated proligand cleaved by TACE that subsequently binds to the EGFR to induce ERK1/2 and other signals required for increased permeability. Surprisingly, because blocking HB-EGF and the EGFR caused increased baseline permeability, these data also suggest a role for the EGFR in the mediation of baseline permeability.
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To further evaluate TGF- and HB-EGF signaling after oxidant stimulation, we carried out phosphoprotein analysis of cell lysates to assess EGFR phosphorylation (Fig. 8) and ERK1/2 activation (Fig. 9) in the presence of the above EGFR, TGF-, and HB-EGF inhibitors when cells were at rest or stimulated with H2O2. The EGFR activation data (Fig. 8) agree closely with the ERK1/2 signaling data (Fig. 9). Figure 8, lane 3 shows cells that were treated with the EGFR inhibitor AG1478; all baseline EGFR phosphorylation was eliminated. The solid bars indicate treatment with H2O2. H2O2 greatly stimulated EGFR phosphorylation (lane 4). Pretreatment with EGFR kinase inhibitor (lane 5) and TACE inhibitor (lane 6) again reduced EGFR tyrosine phosphorylation to below control (baseline) levels seen in lane 2 despite H2O2 stimulation. Thus, as in Fig. 2a, virtually all H2O2-stimulated EGFR phosphorylation was found to be metalloprotease-dependent. Next, specific blocking proteins or Abs to the EGFR or EGFR ligands amphiregulin, HB-EGF, and TGF- were tested for their effect on cell signaling at concentrations equal to those used in the Fig. 7 permeability studies. Blocking Ab to the EGFR significantly inhibited (to below control levels) EGFR phosphorylation stimulated by H2O2 (lane 7). Most important, treatment with blocking Ab to TGF- eliminated EGFR phosphorylation to a level comparable with the effects of EGFR kinase and TACE inhibitors, whereas blocking protein to HB-EGF inhibited by only 40%. We next assessed ERK1/2 phosphorylation in the same cell lysates (Fig. 9). Lane 2 shows that virtually all ERK1/2 activation is due to EGFR signaling and is eliminated with EGFR kinase inhibitor treatment, even in control cells (baseline). H2O2 dramatically stimulated ERK1/2 activation (5-fold) (lane 3), and the EGFR kinase (lane 4) and TACE inhibitors (lane 5) blocked activation of downstream signaling, in this case ERK1/2, to below baseline control values. The blocking Ab to the EGFR (anti-ErbB1) also significantly (–88%) inhibited oxidant-induced ERK1/2 activation but not to the extent (
95%) of the blocking TGF- antibody. Finally, blocking protein to HB-EGF inhibited H2O2-induced ERK1/2 activation, but only by 30%. Blocking Ab to amphiregulin had no effect on EGFR or ERK1/2 phosphorylation at several concentrations (data not shown). Taken together, these permeability and phosphoprotein signaling data strongly support a metalloprotease-dependent EGFR transactivation mechanism regulating permeability of this in vitro model in response to oxidant stress. In addition, the data support a model in which TGF- is the proligand cleaved by TACE to initiate the EGFR-ERK1/2 signaling required for the increase in permeability.
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The EGFR and the EGFR Proligands TGF- and HB-EGF Are Primarily Localized to Peripheral Cell-Cell Junctions. We next sought to use immunofluorescence microscopy to identify the cellular locations of the EGFR and EGFR proligands TGF- and HB-EGF. Figure 10 shows the results from staining unstimulated Caco-2 monolayers on coverslips with primary Ab to the EGFR (ErbB1) followed with FITC secondary Ab (green) as well as Ab to TACE followed by Texas Red secondary Ab. Clearly, the EGFR location is concentrated at the cell periphery in areas of cell-cell adhesion (arrows). No differences were seen after treatment with H2O2 (data not shown). Figure 11, a and b, show representative images resulting from Caco-2 on coverslips with primary Ab to either HB-EGF (Fig. 11a) or TGF- (Fig. 11b) followed by FITC secondary Ab. Again, in both cases, the EGFR proligands are also primarily localized to cell-cell contact zones, which also did not change with oxidant stimulation (data not shown).
We next used immunofluorescent microscopy to identify the location of TACE before and after stimulation with oxidant (Figs. 12, 13, 14). To do this, we treated cells with Ab to the intracellular domain of TACE (+Texas Red 2°Ab) and the tight junction protein ZO-1 (+FITC 2°Ab), either as media-treated control (Fig. 12, a–c, top) cells or cells treated with H2O2 (Fig. 12, d–f, bottom). These images are representative of multiple experiments and images and clearly show several key points. First, note the characteristic deformation of ZO-1 staining in the H2O2-treated (bottom) panel as seen in Fig. 1 that is consistent with increased permeability. The next point is that TACE staining in unstimulated cells is characteristically without pattern and distributed diffusely within each cell as viewed two-dimensionally (arrows). However, in oxidant-stimulated cells, TACE staining consistently exhibits a strong distribution to the cell periphery and forms characteristic "ring-like" structures (arrows). Thus, stimulation with oxidant seems to result in dramatic TACE translocation to the cell-cell contact zones where the EGFR, TGF-, and HB-EGF are already located. The translocation of TACE to colocalize with ZO-1 is shown in the x-z views compiled from deconvoluted z-stacks in Fig. 13. In the Fig. 13 x-z and y-z projections, note the large distribution of TACE (red) below the plane of ZO-1 (green) in control cells (unstimulated), whereas TACE can clearly be seen to colocalize with ZO-1 in the H2O2-stimulated cells. This redistribution of TACE to colocalize with ZO-1 is most clear in the 3-D reconstructions seen in Fig. 14. In Fig. 14, A and B, the TACE (red) pool is clearly located beneath ZO-1 (green) in a virtual bilayer arrangement. Dramatically, in the oxidant-stimulated cells, TACE translocates to colocalize and intersperse with ZO-1, and the bottom pool of TACE disappears (Fig. 14, C and D). In the unstimulated cells, TACE (red) exists predominantly in a pool beneath ZO-1 (green) that is perinuclear as reported by others in different cell types (Soond et al., 2005). Staining for nuclei with 4,6-diamidino-2-phenylindole has been left out of these figures because it obscured the view of TACE staining; however, the perinuclear (bottom) pool of TACE is still clear in the three-dimensional views in Fig. 14, A and B. The bottom TACE pool is especially clear in Fig. 14A (orthogonal, from below), and this clearly becomes intercalated with ZO-1 in Fig. 14C. Taken together, these series of fluorescence microscopic images in Figs. 12, 13, 14 clearly demonstrate movement of TACE to colocalize with ZO-1 in oxidant-stimulated cells consistent with the proposed role for TACE in regulating TJ permeability in IECs.
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and oxidants are widely acknowledged as key proinflammatory factors involved in tissue injury in both ulcerative colitis and Crohn's disease (Podolsky, 2002; Farhadi et al., 2003). Indeed, anti-TNF- (Zeissig et al., 2004) or antioxidant (Keshavarzian et al., 1992) therapies have been shown to be effective in the treatment of IBD. One of the mechanisms underlying the deleterious effects of oxidants and TNF- in IBD is probably increased intestinal permeability (Bruewer et al., 2003; Clayburgh et al., 2004). However, the mechanisms of oxidant- and TNF--induced intestinal hyperpermeability in IBD are not fully understood. It is known that TACE is the metalloprotease required for TNF- cleavage and biological activity (Black et al., 1997). Both TACE and TNF- are up-regulated in active IBD as well as in normal-appearing mucosa of IBD patients (Brynskov et al., 2002; Zeissig et al., 2004). TNF- is also known to stimulate TACE expression in a kind of vicious circle (Black et al., 1997). Our study now provides the novel finding that oxidant-induced barrier hyperpermeability is partly dependent on TACE metalloprotease-mediated transactivation of EGFR signaling. This study provides compelling data to support a new model (oxidant-induced metalloprotease-dependent EGFR transactivation) that could identify a new relationship between oxidants and TNF- in inducing intestinal hyperpermeability and tissue injury in IBD. The key points of this model are summarized in Fig. 15. The model begins with oxidant stress (1), resulting in activation of the metalloprotease TACE (2). Activated TACE then cleaves membrane TGF- to a soluble form (3) that binds to the EGFR. EGFR activation then results in downstream MAPK/ERK1/2 activation (4). ERK1/2 signaling (5) then results in increased intestinal permeability (6) by pathways that have yet to be identified.
Metalloprotease-EGFR signaling has been demonstrated in IECs in response to Substance-P (Koon et al., 2004), and Clostridium difficile toxin (Na et al., 2005) as a regulator of cell proliferation. Previous studies from this and other laboratories have shown a role for the EGFR in regulation of intestinal permeability in response to oxidative stress (Banan et al., 2000b, 2001a; Basuroy et al., 2006). To our knowledge, however, ours is the first demonstration of metalloprotease-EGFR transactivation signaling in the regulation of intestinal barrier function by any stimulus and, in this case, in response to oxidative stress.
Several of our results strongly support TACE as the primary metalloprotease involved. First is potent inhibition of oxidant-induced intestinal hyperpermeability by GM6001 as well as by the TACE-specific inhibitor TAPI-2. TACE is not an MMP but is instead a member of the ADAM family of membrane metalloproteases. Although also capable of inhibiting MMP type metalloproteases, GM6001 has been shown by others to inhibit TACE but not necessarily other ADAM metalloproteases (Santiskulvong and Rozengurt, 2003). In contrast, TAPI-2 was originally identified by its ability to inhibit TACE but not MMPs; however, its specificity against other ADAM metalloproteases in the TACE family is not well defined (Mohler et al., 1994). Second, both the EGFR and TACE inhibitors also block oxidant-induced displacement in ZO-1 staining that is characteristic of increased permeability (Banan et al., 2001a; Basuroy et al., 2006). Third, RT-PCR and Western-blotting data show that Caco-2 cells in our model express abundant TACE, including the key 90-kDa activated form (Black et al., 1997). Fourth, gene-specific SiRNA knockdown studies of TACE reveal that TACE is the predominant metalloprotease mediating H2O2-induced ERK1/2 activation required for increased permeability in our model. Fifth and finally, our images also significantly show that with oxidant stimulation TACE is translocated to cell-cell junction areas and colocalizes with ZO-1 in proximity with the proposed ligand/receptor in this case: TGF-/EGFR. These translocation images support a model proposed by others for translocation of TACE with specific proligand substrates as a key regulatory mechanism of TACE specificity (Werb and Yibing, 1998). Our TGF- permeability and signaling blocking data clearly support that TGF- is the principal EGFR proligand cleaved by TACE upon H2O2 stimulation, and these data are further supported by the dramatic peripheral colocalization of TACE-TGF- in oxidant-stimulated cells. This is consistent with TACE being the major sheddase for TGF- cleavage in EGFR transactivation in other cell types (Blobel, 2005).
Our data show the mechanism for increased permeability due to H2O2 transactivation of EGFR signaling is mediated in part by MAPK signaling, specifically ERK1/2. Thus, another concept that emerges from this study is that ERK1/2 activation seems to be one critical pathway mediating intestinal hyperpermeability in response to oxidants through TACE-EGFR signaling. This is supported by the comparative permeability and signaling data since only the EGFR, TACE, and ERK1/2 inhibitors showed similar protection against increased permeability, and these three inhibitors exhibited the most significant inhibition of both EGFR and ERK1/2 activation by oxidants. These data agrees with that of others showing tyrosine kinase and Src inhibitors block H2O2-induced permeability in Caco-2 monolayers (Basuroy et al., 2003). This result also agrees with data from others showing that both TGF--induced hyperpermeability in Madin-Darby canine kidney cells (Chen et al., 2000) and H2O2-induced endothelial hyperpermeability (Fischer et al., 2005) can be blocked with the same ERK1/2 inhibitor. However, these data seem to differ from other studies showing ERK1/2 activation is required for EGF protection of Caco-2 monolayers from oxidant stress-induced hyperpermeability. The reason for this difference is not clear. One possibility is that the study referred to used very low concentrations of H2O2 (20 µM) that do not result in ERK1/2 activation in their data or in our laboratory. Therefore the mechanism and role of ERK1/2 may be different. The concentration of H2O2 used in the present study is consistent with the 200 to 500 µM concentration used by other investigators in the initial discovery of oxidant-induced EGFR transactivation (Prenzel et al., 1999) as well as more recent intestinal epithelial cell studies by others (Song et al., 2006). We have stated here and in previous studies this concentration does not affect cell viability (Banan et al., 2000a,b).
Another key difference is the point we refer to as the "EGF-EGFR paradox." The paradox is as follows. Preincubation with EGFR ligands EGF or TGF- protects against oxidant-induced permeability. However, paradoxically, EGFR signaling seems to be required for oxidant-induced permeability. The mechanism for this at present is unknown but has precedent in many of the preconditioning models in cell biology in which preconditioning with a stimulus "protects" against subsequent challenge with that stimulus (e.g., heat shock, hypoxia). In fact, ERK1/2 have been shown to be important in oxidant preconditioning in other cell types (Hung et al., 2003). Although our own studies have shown previously that PLC- (Banan et al., 2001a) and PKC-1 (Banan et al., 2002) are also critical for this EGF preconditioning protection, the complete mechanism for this paradox remains to be identified in future studies but seems to also require ERK1/2 signaling (Basuroy et al., 2006). Taken together, it is clear that EGFR signaling as well as ERK1/2 signaling play critical roles in oxidant-induced intestinal permeability and protection.
Our data showing that ERK inhibitors block oxidant-induced hyperpermeability agrees with studies with MDCK and endothelial cells. Therefore, key downstream targets of ERK seem to be critical in the regulation of paracellular permeability in many cell types. One possible candidate suggested in other studies is myosin light-chain kinase (MLCK) (Clayburgh et al., 2004). ERK1/2 directly phosphorylates MLCK (Nguyen et al., 1999), and MLCK activation of myosin light chain is one key mechanism regulating intestinal permeability and may play a role in IBD (Clayburgh et al., 2004). In addition, ERK1/2 can be immunoprecipitated with the TJ protein occludin in oxidant-treated IECs (Basuroy et al., 2006), suggesting phosphorylation of occludin by ERK1/2 as another mechanism for ERK1/2 regulation of TJ permeability. Finally, ERK1/2 have been shown to regulate TACE activation and phosphorylate TACE directly (Soond et al., 2005). Further studies will be needed to identify the specific role(s) for ERK1/2 in TACE/EGFR regulation of intestinal permeability.
In summary, we propose a new model in which metalloprotease-dependent EGFR transactivation in response to oxidant stress is one mechanism for intestinal hyperpermeability and tissue injury in IBD. In this model, the metalloprotease TACE plays a key role by cleavage of EGFR ligands and TNF-. Indeed, metalloprotease inhibitors of TACE eliminate inflammation in animal models of IBD (Colon et al., 2001). Our permeability data with the CRM197-specific blocking protein for HB-EGF and our microscopy data showing it localized to cell-cell junction zones support a model in which HB-EGF signaling regulates baseline barrier integrity. This could occur possibly via juxtacrine interaction by HB-EGF proligands with EGFR on neighboring cells, which is known to inhibit mitosis (Singh et al., 2004).
Thus, our findings could have far-reaching implications for a better understanding of IBD pathogenesis using a new model of intestinal hyperpermeability. These data also identify potential targets for developing new therapies for IBD such as specific inhibitors of TACE-EGFR-initiated signaling and ERK1/2 inhibitors. Further in vivo animal studies are currently underway in our laboratory to determine whether modulation of the metalloprotease-dependent EGFR transactivation pathway can be successfully and safely targeted in IBD therapy.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: IBD, inflammatory bowel disease; TNF, tumor necrosis factor; TJ, tight junction; IEC, intestinal epithelial cell; EGFR, epidermal growth factor receptor; PKC, protein kinase C; EGF, epidermal growth factor; TGF, transforming growth factor; TACE, tumor necrosis factor--converting enzyme; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; TAPI-2, N-(R)-[2-(hydroxyaminocarbonyl)methyl]-4-methylpentanoyl-L-t-butyl-alanyl-L-alanine, 2-aminoethyl amide, TNF- protease inhibitor-2; GM6001, Ilomastat or N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide; AG1478, 4-(3-chloroanilino)-6,7-dimethoxyquinazoline; GW2974, N4-(1-benzyl-1H-indazol-5-yl)-N 6,N 6-dimethyl-pyrido[3,4-D]pyrimidine-4,6-diamine; PD98059, 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene ethanolate; p38, 38-kDa protein (MAPK); SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole; JNK, jun kinase; SP600125, 1,9-pyrazoloanthrone anthrapyrazolone; LY29004, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; TAPI, TNF- protease inhibitor; Ab, antibody; HB-EGF, heparin binding epidermal growth factor; AKT, protein kinase B; DMEM, Dulbecco's modified Eagle's medium; FSA, fluorescein-5-(and-6)-sulfonic acid trisodium salt; FITC, fluorescein isothiocyanate; RT, reverse transcription; PCR, polymerase chain reaction; SiRNA, short inhibitory RNA; MMP, matrix metalloproteinase; DMSO, dimethyl sulfoxide; MLCK, myosin light chain kinase; ADAM, A disintegrin and metalloproteinase; MMPi, matrix metalloprotease inhibitor.
Address correspondence to: Dr. Christopher B. Forsyth, Rush University Medical Center, Department of Internal Medicine, Section of Gastroenterology, 1725 W. Harrison, Suite 206, Chicago, IL 60612. E-mail: christopher_b_forsyth{at}rush.edu
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References |
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|---|
Banan A, Choudhary S, Zhang Y, and Keshavarzian A (2000a) Role of the microtubule cytoskeleton in protection by epidermal growth factor and transforming growth factor- against oxidant-induced barrier disruption in a human colonic cell line. Free Radic Biol Med 28: 727–738.[CrossRef][Medline]
Banan A, Fields JZ, Farhadi A, Talmadge DA, Zhang Y, and Keshavarzian A (2002) The beta 1 isoform of protein kinase C mediates the protective effects of epidermal growth factor on the dynamic assembly of F-actin cytoskeleton and normalization of calcium homeostasis in human colonic cells. J Pharmacol Exp Ther 301: 852–866.
Banan A, Fields JZ, Zhang Y, and Keshavarzian A (2001a) Phospholipase C-gamma inhibition prevents EGF protection of intestinal cytoskeleton and barrier against oxidants. Am J Physiol 281: G412–G423.
Banan A, Zhang Y, Fields JZ, and Keshavarzian A (2001b) Key role of PKC and calcium homeostasis in epidermal growth factor-induced protection of the microtubule cytoskeleton and intestinal barrier against oxidant injury. Am J Physiol 280: G828–G843.
Banan A, Zhang Y, Losurdo J, and Keshavarzian A (2000b) Carbonylation and disassembly of the F-actin cytoskeleton in oxidant-induced barrier dysfunction and its prevention by epidermal growth factor and transforming growth factor- in a human intestinal cell line. Gut 46: 830–837.
Banan A, Zhang LJ, Shaikh M, Fields JZ, Choudhary S, Forsyth CB, Farhadi A, and Keshavarzian A (2005) Theta isoform of protein kinase C alters barrier function in intestinal epithelium through modulation of distinct claudin isotypes: a novel mechanism for regulation of permeability J Pharmacol Exp Ther 313: 962–982.
Banan A, Zhang LJ, Shaikh M, Fields JZ, Farhadi A, and Keshavarzian A (2004) Novel effect of NF-kB activation: carbonylation and nitration injury to cytoskeleton and disruption of monolayer in intestinal epithelium. Am J Physiol 287: C1139–C1151.
Basuroy S, Seth A, Elias B, Anjaparavanda P, Naren A, and Rao R (2006) MAP kinase interacts with occludin and mediates EGF-induced prevention of tight junction disruption by hydrogen peroxide. Biochem J 393: 69–77.[CrossRef][Medline]
Basuroy S, Seth P, Kuppuswamy D, Balasubramanian S, Ray RM, and Rao RK (2003) Expression of kinase-inactive c-Src delays oxidative stress-induced disassembly and accelerates calcium-mediated reassembly of tight junctions in the Caco-2 cell monolayer. J Biol Chem 278: 11916–11924.
Black RA, Rauch CT, Kozlosky CJ, Peschon JJ, Slack JL, Wolfson MF, Castner BJ, Stocking KL, Reddy P, Srinivasan S, et al. (1997) A metalloproteinase disintegrin that releases tumor-necrosis factor-alpha from cells. Nature (Lond) 385: 729–733.[CrossRef][Medline]
Blobel CP (2005) ADAMs: key components in EGFR signaling and development. Nat Rev Mol Cell Biol 6: 32–43.[CrossRef][Medline]
Bruewer M, Luegering A, Kucharzik T, Parkos CA, Madara JL, Hopkins AM, and Nusrat A (2003) Proinflammatory cytokines disrupt epithelial barrier function by apoptosis-independent mechanisms. J Immunol 171: 6164–6172.
Brynskov J, Foegh P, Pederson G, Ellervik C, Kirkegaard T, Bingham A, and Saermark T (2002) Tumor necrosis factor converting enzyme (TACE) activity in the colonic mucosa of patients with inflammatory bowel disease. Gut 51: 37–43.
Chen Y, Lu Q, Schneeberger EE, and Goodenough DA (2000) Restoration of tight junction structure and barrier function by down-regulation of the mitogen-activated protein kinase pathway in Ras-transformed Madin-Darby canine kidney cells. Mol Biol Cell 11: 849–862.
Clayburgh DR, Shen L, and Turner JR (2004) A porous defense: the leaky epithelial barrier in intestinal disease. Lab Investig 84: 282–291.[CrossRef][Medline]
Colon AM, Menchen LA, Hurtado O, De Cristobal J, Lizasoain I, Leza JC, Lorenzo P, and Moro MA (2001) Implication of TNF-alpha convertase (TACE/ADAM17) in inducible nitric oxide synthase expression and inflammation in an experimental model of colitis. Cytokine 16: 220–226.[CrossRef][Medline]
Farhadi A, Banan A, Fields J, and Keshavarzian A (2003) Intestinal barrier: an interface between health and disease. J Gastroenterol Hepatol 18: 479–487.[CrossRef][Medline]
Fischer OM, Hart S, Gschwind A, Prenzel N, and Ullrich A (2004) Oxidative and osmotic stress signaling in tumor cells is mediated by ADAM proteases and heparin-binding epidermal growth factor. Mol Cell Biol 24: 5172–5183.
Fischer S, Wiesnet M, Renz D, and Schaper W (2005) H2O2 induces paracellular permeability of porcine brain-derived microvascular endothelial cells by activation of the p44/42 MAP kinase pathway. Eur J Cell Biol 84: 687–697.[CrossRef][Medline]
Forsyth CB, Pulai J, and Loeser RF (2002) Fibronectin fragments and blocking antibodies to alpha2beta1 and alpha5beta1 integrins stimulate mitogen-activated protein kinase signaling and increase collagenase 3 (matrix metalloproteinase 13) production by human articular chondrocytes. Arthritis Rheum 46: 2368–2376.[CrossRef][Medline]
Hung CC, Ichimura T, Stevens JL, and Bonventre JV (2003) Protection of renal cells against oxidative injury by endoplasmic reticulum stress preconditioning is mediated by ERK1/2 activation. J Biol Chem 278: 29317–29326.
Keshavarzian A, Haydek J, Zabihi R, Doria M, D'Astice M, and Sorenson JR (1992) Agents capable of eliminating reactive oxygen species, catalase, WR-2721, or Cu(II)2(3,5-DIPS)4 decrease experimental colitis. Dig Dis Sci 37: 1866–1873.[CrossRef][Medline]
Keshavarzian A, Morgan G, Sedgu S, Gordon JH, and Doria M (1990) Role of reactive oxygen metabolites in experimental colitis. Gut 31: 786–790.
Koon HW, Zhao D, Na X, Moyer MP, and Pothoulakis C (2004) Metalloproteinases and transforming growth factor-alpha mediate substance P-induced mitogen-activated protein kinase activation and proliferation in human colonocytes. J Biol Chem 279: 45519–45527.
Mohler KM, Sleath PR, Fitzner JN, Cerretti DP, Alderson M, Kerwar SS, Torrance DS, Otten-Evans C, Greenstreet T, Weerawarna K, et al. (1994) Protection against a lethal dose of endotoxin by an inhibitor of tumor necrosis factor processing. Nature (Lond) 370: 218–220.[CrossRef][Medline]
Na X, Zhao D, Koon HW, Kim H, Husmark J, Moyer MP, Pothoulakis C, and LaMont JT (2005) Clostridium difficile toxin B activates the EGF receptor and ERK/MAP kinase pathway in human colonocytes. Gastroenterology 128: 1002–1011.[CrossRef][Medline]
Nguyen DH, Catling AD, Webb DJ, Sankovic M, Wlker LA, Somlyo AV, Weber MJ, and Gonias SL (1999) Myosin light chain kinase functions downstream of Ras/ERK to promote migration of urokinase-type plasminogen activator-stimulated cells in an integrin selective manner. J Cell Biol 146: 149–164.
Podolsky DK (2002) Inflammatory bowel disease. N Engl J Med 347: 417–429.
Prenzel N, Zwick E, Daub H, Leserer M, Abraham R, Wallasch C, and Ullrich A (1999) EGF receptor transactivation by G-protein-coupled receptors requires metalloproteinase cleavage of proHB-EGF. Nature (Lond) 402: 884–888.[Medline]
Sanders SE, Madara JL, McGuirk DK, Gelman DS, and Colgan SP (1995) Assessment of inflammatory events in epithelial permeability: a rapid screening method using fluorescein dextrans. Epithelial Cell Biol 4: 25–34.[Medline]
Santiskulvong C and Rozengurt E (2003) Galardin (GM6001), a broad-spectrum matrix metalloproteinase inhibitor, blocks bombesin- and LPA-induced EGF receptor transactivation and DNA synthesis in rat-1 cells. Exp Cell Res 290: 437–446.[CrossRef][Medline]
Schafer B, Gschwind A, and Ullrich A (2004) Multiple G-protein-coupled receptor signals converge on the epidermal growth factor receptor to promote migration and invasion. Oncogene 23: 991–999.[CrossRef][Medline]
Schlondorff J, Becherer JD, and Blobel CP (2000) Intracellular maturation and localization of the tumor necrosis factor alpha convertase. Biochem J 347: 131–138.
Singh AB, Tsukada T, Zent R, and Harris RC (2004) Membrane associated HB-EGF modulates HGF-induced cellular responses in MDCK cells. J Cell Sci 117: 1365–1379.
Song J, Li J, Lulla A, Evers BM, and Chung DH (2006) Protein kinase D protects against oxidative stress-induced intestinal epithelial cell injury via Rho/ROK/PKC-delta pathway activation. Am J Physiol 290: 1469–1476.
Soond SM, Everson B, Riches DWH, and Murphy G (2005) ERK-mediated phosphorylation of Thr735 in TNF-alpha converting enzyme and its potential role in TACE protein trafficking. J Cell Sci 118: 2371–2380.
Werb Z and Yibing Y (1998) A cellular striptease act. Science (Wash DC) 282: 1279–1280.
Zeissig S, Bojarski C, Buergel N, Mankertz J, Zeitz M, Fromm M, and Schulzke JD (2004) Downregulation of epithelial apoptosis and barrier repair in active Crohn's disease by tumor necrosis factor-alpha antibody treatment. Gut 53: 1295–1302.
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