Tumor necrosis factor regulates intestinal epithelial cell migration by receptor-dependent mechanisms

Julissa Corredor1, Fang Yan1, Christopher C. Shen1, Wei Tong1, Sutha K. John1, Guinn Wilson1, Robert Whitehead2,3, and D. Brent Polk1,2

Departments of 1 Pediatrics, 2 Cell and Developmental Biology, and 3 Medicine, Division of Gastroenterology and Nutrition, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2576


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Altered mucosal integrity and increased cytokine production, including tumor necrosis factor (TNF), are the hallmarks of inflammatory bowel disease (IBD). In this study, we addressed the role of TNF receptors (TNFR) on intestinal epithelial cell migration in an in vitro wound closure model. With mouse TNFR1 or TNFR2 knockout intestinal epithelial cells, gene transfection, and pharmacological inhibitors, we show a concentration-dependent receptor-mediated regulation of intestinal cell migration by TNF. A physiological TNF level (1 ng/ml) enhances migration through TNFR2, whereas a pathological level (100 ng/ml) inhibits wound closure through TNFR1. Increased rate of wound closure by TNFR2 or inhibition by TNFR1 cannot be explained by either increased proliferation or apoptosis, respectively. Furthermore, inhibiting Src tyrosine kinase decreases TNF-induced focal adhesion kinase (FAK) tyrosine phosphorylation and cellular migration. We therefore conclude that TNFR2 activates a novel Src-regulated pathway involving FAK tyrosine phosphorylation that enhances migration of intestinal epithelial cells.

intestinal restitution; Src; focal adhesion kinase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE SINGLE LAYER of epithelial cells lining the intestine forms an important barrier to a broad spectrum of noxious and immunogenic substances within the lumen. Continued cellular migration along the intestinal crypt-villus axis is necessary for maintenance of this layer through normal cell turnover, mucosal wound healing, and prevention of disease (18). This process is highly regulated by growth factors, cytokines, and matrix metalloproteinases (36, 51). An imbalance between mediators of the inflammatory process with subsequent altered mucosal reepithelialization has been described in individuals with inflammatory bowel disease (IBD), specifically an excess of proinflammatory mediators such as tumor necrosis factor (TNF), interleukin (IL)-8, and IL-12 and/or deficit of anti-inflammatory mediators such as IL-10 (3, 9, 25, 33, 34, 45). Conversely, animals in a germ-free environment (19) or on an elemental diet (10, 30) develop intestinal atrophy. Therefore, we (22) and others (19) have hypothesized that interactions between the intestinal epithelial cell and the contents of the gastrointestinal tract including commensal bacteria, pathogenic bacteria in low numbers, or other antigens may promote a healthy mucosal barrier through production of physiological cytokine levels.

TNF is a proinflammatory cytokine known to play a key role in the pathogenesis of IBD (7, 40, 48). TNF is expressed as a 26-kDa transmembrane precursor that is cleaved into an active soluble 17-kDa polypeptide, which aggregates into homotrimeric complexes (4). It exerts biological action through two distinct cell surface receptors, TNF receptor (TNFR)1 (55 kDa) and TNFR2 (75 kDa) (17, 28). TNF has a fivefold higher affinity for TNFR2 than TNFR1 in mouse cells, suggesting preferential ligation of TNFR2 at physiological TNF concentrations (49, 52). The two receptors mediate opposing effects on intestinal epithelial cell proliferation (22). TNFR1 induces growth arrest through sustained mitogen-activated protein kinase activity (23), whereas TNF stimulates proliferation via TNFR2 (22). Other cell systems show similar receptor-dependent opposing biological effects of TNF. For example, TNFR1 and TNFR2 mediate differing effects on osteoclastogenesis (1) and endothelial permeability (11).

Although there is clear evidence linking TNF to the pathogenesis of IBD, little is known about the effects of TNF on mucosal injury and repair. We have hypothesized that physiological levels of TNF play a role in maintaining an intact intestinal mucosal barrier. For instance, mice lacking TNFR1 receptor do not develop Peyer's patches and have defective germinal center formation (29, 32). Absence of TNFR2 correlates with increased sensitivity to bacterial pathogens (39) and decreased sensitivity to polysaccharide antigens in the intestine and colon (27). However, we are unaware of studies regarding the functional role of TNFR1 or TNFR2 on intestinal epithelial restitution. Therefore, we studied the effect of TNF on intestinal cell migration in a wound closure model. We describe concentration-dependent receptor-mediated regulation of intestinal cell migration by TNF. These findings are consistent with a model in which physiological levels of TNF enhance intestinal cell migration through TNFR2 and pathological levels inhibit epithelial wound closure through TNFR1. These data have potential implications for understanding altered reepithelialization of mucosal ulceration in IBD.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. 5-Bromo-2'-deoxyuridine (BrdU) Labeling and Detection Kit I was purchased from Roche Applied Science (Indianapolis, IN). Anti-focal adhesion kinase (FAK) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-FAK-PY576 antibody was a gift of Steven Hanks (Vanderbilt University, Nashville, TN). Anti-phospho Y416-Src antibody was from Cell Signaling Technology (Beverly, MA). Anti-Src antibody was from Upstate Biotechnology (Lake Placid, NY). Goat anti-human TNFR1 agonist antibody was purchased from R&D Systems (Minneapolis, MN). ApopTag Fluorescein In Situ Apoptosis Detection Kit was from Intergen (Purchase, NY). Human recombinant epidermal growth factor (EGF) was provided by Carlos George-Nascimento (Chiron, Emeryville, CA). Human recombinant hepatocyte growth factor (HGF) was purchased from Collaborative Medical Products (Bedford, MA). Monoclonal hamster anti-mouse TNFR1 antagonist was obtained from Genzyme Diagnostics (Cambridge, MA). Murine recombinant TNF was purchased from Pepro Tech (Rocky Hill, NJ). Rabbit anti-human TNFR2 polyclonal antibodies BGL 74935 (directed against both extracellular and intracellular domains including the carboxy terminus) and SMA 73932 (directed against the extracellular domain) were gifts of Renu Heller (Stanford University, Stanford, CA). Rat tail collagen type I was purchased from Collaborative Medical Products. Recombinant murine interferon (IFN)-gamma and murine fibronectin were from Life Technologies (Gaithersburg, MD). The Src tyrosine kinase-specific inhibitor PD-161430 was a gift of David Fry (Parke-Davis Pharmaceutical Research, Ann Arbor, MI). PP1 and PP2 were from Biomol (Plymouth Meeting, PA).

Cell line preparation. The conditionally immortalized young adult mouse colon (YAMC) and mouse small intestine epithelial (MSIE) cell lines were established from the colonic and small intestinal mucosa of the H-2Kb-tsA58 transgenic mouse (Immortomouse) expressing a heat-labile simian virus (SV)40 large T antigen with an IFN-gamma -inducible promoter as previously described (22, 43, 56). TNFR1-/- and TNFR2-/- mouse colon epithelial (MCER1-/- and MCER2-/-, respectively) cell lines were prepared by mating the transgenic knockout mice with the Immortomouse (Charles River Laboratories, Wilmington, MA). Mice carrying the SV40 gene were identified by PCR as previously described (56). The mice that were SV40 positive were then tested for the presence of the transgene. Mice carrying both genes were mated to regain the homozygous deletion of the transgene.

Young adult mice with a homozygous deletion of the transgene and carrying the SV40 gene were killed. The colon was removed, opened, washed and incubated in 0.04% sodium hypochlorite for 15 min at room temperature. The colon was then washed in phosphate-buffered saline (PBS) and incubated in 3 mM EDTA plus 0.5 mM DTT in PBS for 90 min at room temperature. The colon was transferred to a screw-capped tube in 15 ml of PBS and shaken vigorously to liberate the crypts from the mucosa. These crypts were then concentrated by centrifugation at 400 rpm for 5 min. Next, the crypts were resuspended in RPMI 1640 plus 5% FBS, 10% conditioned medium from the LIM1863 colon carcinoma cell line, and 20 U/ml mouse IFN-gamma as previously described (56). The crypt suspension was plated into the wells of collagen 1-coated 24-well tissue culture dishes with a minimal volume of medium in each well and incubated at 33°C. After 48-h incubation, 1 ml of medium was added to each well. The medium was changed twice a week. When cell growth was evident the cells were passaged with trypsin-EDTA solution.

The epithelial phenotype of the cells was confirmed by cytokeratin staining. The cells were cultured on eight-well LabTek slides until they were 80% confluent. The slides were then washed in PBS, fixed in cold acetone for 15 min, and air dried. Next, cells were incubated with a monoclonal antibody to keratin 18 (LE61, a kind gift of Dr. E. B. Lane, Cancer Research Center, Cell Structure Research Group, University of Dundee School of Life Science, Dundee, UK). The presence of a characteristic basket weave staining pattern in the cytoplasm was considered evidence of the presence of the keratin fibers indicative of epithelial cells.

Cell culture. Cells were maintained in RPMI 1640 medium with 5% FBS and 5 U/ml murine IFN-gamma and grown under permissive conditions at 33°C with 5% CO2 (22). Before all experiments, cells were plated onto fibronectin-coated (1 µg/ml) surfaces (43) and then transferred to 37°C under nonpermissive conditions with 0.5% FBS IFN-gamma -free medium for 24 h.

Migration assays. Confluent cells were trypsinized and plated (1.5 × 105 cells) on 35-mm culture dishes coated with fibronectin. After cell attachment (2 h), the medium was changed to 0.5% FBS IFN-gamma -free medium and incubated overnight at 37°C. The medium was then aspirated, and seven to nine circular 1-mm cell-free areas were created with a stabilized rotating silicone tip, using a method similar to that described by Watanabe and colleagues (54). Immediately after wounding, the cells were washed twice with PBS, with culture medium replaced with the indicated factors in the presence or absence of agonist, antagonist antibodies, or pharmacological inhibitors, and then cultured up to 24 h. The surface areas of the initial wound (set at 100%) and at the end of treatment were recorded by time-lapse video microscopy and measured by BioQuant Image Analysis software (Nashville, TN).

Cellular transfections. Transient transfection of pRK7-p75 or empty vector (gift of Mike Rothe, Tularik, South San Francisco, CA) was performed on p75-/- YAMC cells with 1.5 µg of DNA and reagents provided in the Lipofectamine Plus Reagent kit (GIBCO BRL, Grand Island, NY) following manufacturer's guidelines. After transfection for 24 h, cells were incubated in RPMI medium with 0.5% FBS at 37°C for 6 h before experiments. p75 expression was detected with the PCR method. The forward primer sequence and its reverse complement were 5'-ATGGCGCCCGTCGCCGTCTGGGCCGCG-3' and 5'-CAGGCGAGTGAGGCACCTTGGCTTC-3', respectively. PCR products were resolved and visualized on 2% agarose gel.

Proliferation assay. Wounded and nonwounded YAMC cells cultured on fibronectin-coated chamber slides were treated with TNF or EGF for 8 or 24 h. At the end of treatment, cell proliferation was detected by BrdU labeling. Cells were incubated with 10 µM BrdU in cell culture medium for 30 min at 37°C, and then BrdU-incorporated cells were detected by mouse anti-BrdU antibody and fluorescein-conjugated anti-mouse IgG and observed by fluorescence microscopy. Proliferation rate was determined by counting at least 200 cells in randomly chosen fields and expressing BrdU-positive cells as a percentage of the total number of cells counted. Proliferation around wound sites was determined by counting all cells in the circumference to ~20 cell layers from the wound edge.

Apoptosis assay. YAMC cells were cultured on fibronectin-coated chamber slides and then wounded or not and treated with TNF or EGF for 24 h. At the end of treatment, cells were fixed with 1% paraformaldehyde in PBS. Apoptotic cells were detected by using terminal deoxynucleotidyl transferase [TdT; also termed TdT-mediated dUTP nick end labeling (TUNEL)] and labeled with FITC-conjugated anti-digoxigenin. Slides were then dehydrated and mounted with Vectashield mounting medium. Slides were counterstained with 4,6-diamidino-2-phenylindole (DAPI) by using 1 µg/ml DAPI in mounting medium. The cells were observed by fluorescence microscopy. The percentage of cells undergoing apoptosis was then determined as described for proliferation assays.

Preparation of cellular lysates. After cells were treated with EGF, TNF, or HGF in the presence or absence of TNFR1 agonist, TNFR1 antagonist, or TNFR2 antagonist antibodies for indicated times, cells were rinsed twice with ice-cold PBS and then scraped into cell lysis buffer containing 1% Triton X-100, 20 mM HEPES (pH 7.5), 1 mM orthovanadate, 50 mM glycerol phosphate, 1 mM sodium pyrophosphate, 10 mg/ml leupeptin, 10 mg/ml aprotinin, 18 mg/ml PMSF, and incubated for 30 min on ice. The lysates were centrifuged (14,000 g, 15 min) at 4°C, and the protein concentration of the Triton-soluble fraction was determined with a DC protein assay (Bio-Rad Laboratories, Hercules, CA). The Triton-soluble fraction was collected for immunoprecipitation or mixed with Laemmli sample buffer for SDS-PAGE and Western blot analysis with anti-phospho-Src or anti-Src antibodies.

Immunoprecipitation. Triton-soluble lysates were incubated with anti-FAK antibody at 4°C for 2 h. Recombinant protein A Sepharose conjugate was then added to the mixture and incubated overnight at 4°C. The immunoprecipitates were recovered by centrifugation (14,000 g, 1 min) and washed three times with ice-cold modified RIPA buffer (1% Triton X-100, 1% deoxycholate, 0.1% SDS, 1 mM EDTA, 10 mM Tris, pH 8.5, and 150 mM NaCl), resuspended in Laemmli sample buffer, and denatured for 5 min at 95°C for Western blot analysis with anti-FAK-PY576 or anti-FAK antibodies.

Statistical analysis. The statistical significance of differences between mean values was assessed with paired Student's t-test analysis. The level of statistical significance was set at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

TNF regulates intestinal cell migration in a concentration-dependent manner. Two of the hallmarks of IBD include altered mucosal integrity and increased production of cytokines such as TNF. Therefore, we tested the effect of TNF on migration in an intestinal epithelial wound closure model. Migration assays were performed on cells plated as a monolayer on mouse fibronectin and then treated with EGF or TNF after wounding. As expected (41), EGF increases the rate of wound closure (Fig. 1A). Interestingly, TNF at lower concentrations (1 ng/ml) increases wound closure at rates comparable to EGF. However, higher concentrations of TNF (10 and 100 ng/ml) block migration and completely inhibit EGF-stimulated wound closure. Representative wounds are shown for YAMC cells at 8 h in Fig. 1B. With Giemsa staining we observed that the rotating silicone disk removes cells without removing the underlying extracellular matrix (data not shown).


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Fig. 1.   Tumor necrosis factor (TNF) regulates migration in a concentration-dependent manner. Young adult mouse colon (YAMC) cells were plated at confluence on mouse fibronectin and cultured under nonpermissive conditions overnight before a circular "wound" cell-free area of ~1-mm diameter was created. Photomicrographs were taken of each site immediately after wounding, and then epidermal growth factor (EGF; 10 ng/ml) and/or TNF at the indicated concentrations were added to the culture media. Nine wounds were studied for each treatment group. The change in surface area for each wound was compared with the original size (set at 100%) at various periods of time afterwards as described in MATERIALS AND METHODS. The % of original surface area remaining 8 h after wounding is shown in A. Representative paired wounds are shown at 0 and 8 h in B. After wounding and treatment, cells were labeled with 5-bromo-2'-deoxyuridine (BrdU) and detected with anti-BrdU antibody and cell proliferation rates in wounded and nonwounded areas were determined as described in MATERIALS AND METHODS (C). Cells were fixed for TdT-mediated dUTP nick end labeling (TUNEL) staining with apoptotic nuclei labeled with FITC and 4',6-diamidino-2-phenylindole (DAPI) staining (D). * , Wounded area. Cells treated with TNF (100 ng/ml) for 6 h and 1-h pretreatment with wortmannin (100 nM) were a positive control for positive apoptotic staining. FITC- and DAPI-labeled images were taken from the same field. All experiments were performed in triplicate. Bars indicate means ± SD. *P < 0.05 vs. control; psi P < 0.005 vs. TNF (1 ng/ml); @P < 0.002 vs. EGF treatment.

Because cellular proliferation is part of the repair process after intestinal injury (12), we investigated the role of proliferation in our wound closure assay with BrdU labeling. Surprisingly, during wound closure, the proliferation rate decreases around the wound margin compared with the nonwounded area in both TNF (1 ng/ml)- and EGF-treated YAMC cells (Fig. 1C) and MSIE cells (data not shown). Therefore, migration is the major factor contributing to wound closure in this assay. TNF and other cytokines induce intestinal epithelial apoptosis in vivo (31); therefore, we investigated the role of apoptosis in the delay of wound closure caused by high-dose TNF. No apoptosis is detected in either wounded (Fig. 1D) or nonwounded (data not shown) cells treated with EGF and low or high concentrations of TNF for up to 24 h. These results are consistent with our previous finding (59) that high concentrations of TNF initiate antiapoptotic signals preventing cell death, unless perturbed by pharmacological inhibition as shown with wortmannin to block the phosphatidylinositol 3-kinase/Akt pathway (Fig. 1D).

TNF inhibition of cellular migration is mediated by TNFR1. Because we have shown (22) expression of two TNF receptors on intestinal epithelial cells, we asked whether these concentration-dependent effects of TNF on cell migration might be differentially mediated by the two receptors. With the wound closure model, cells were studied in the presence or absence of antagonist antibodies to TNFR1 or TNFR2. The TNFR1 agonist antibody causes inhibition of EGF-stimulated migration, similar to higher concentrations of TNF (Fig. 2A), whereas antagonist antibodies to TNFR1 inhibit this TNF effect. Furthermore, antagonist antibody to TNFR2 does not alter the inhibition of EGF-stimulated migration by TNF. Wounds in YAMC or MSIE cells treated with either EGF or TNF (1 ng/ml) are closed by 24 h, preventing relative comparison between treatments at this time point in either cell line. To further test our hypothesis that activation of TNFR1 inhibits EGF-stimulated cell migration we developed mouse colon cell lines lacking TNFR1 (MCER1-/-). These cells show loss of the inhibitory effect of higher TNF concentrations on wound closure (Fig. 2B). However, they retain stimulation of migration by lower TNF concentrations, EGF, or HGF.


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Fig. 2.   TNF receptor (TNFR)1 inhibits intestinal cell migration. Mouse small intestine epithelial (MSIE) cells were prepared and wounded as in Fig. 1. Cells were treated with EGF and/or TNF at the indicated concentrations in the presence or absence of agonist or antagonist antibodies to TNFR1 or TNFR2, as indicated. The relative change 8 h after wounding is shown in A. Confluent TNFR1-/- mouse colon epithelial (MCER1-/-) cell monolayers were wounded and then cultured in the presence of TNF, EGF, or hepatocyte growth factor (HGF). The relative change 24 h after wounding is shown in B. Bars indicate means ± SD. *P < 0.0001 vs. control; @P < 0.001 vs. EGF treatment; psi P < 0.02 vs. TNF (1 ng/ml).

Enhanced migration is mediated through TNFR2. Our finding that loss of TNFR1 does not prevent TNF-stimulated migration (Fig. 2B) suggests that this effect is mediated through TNFR2. To test this hypothesis we first used antagonist TNFR2 antibodies in the presence of low-dose TNF. Blocking antibody to TNFR2 completely inhibits TNF-stimulated migration but not EGF-stimulated wound closure (Fig. 3A). In contrast, blocking TNFR1 had no effect on TNF promoting intestinal cell migration. We developed TNFR2 -/- mouse colon epithelial (MCER2-/-) cell lines to verify the role of TNFR2 in stimulating cell migration. MCER2-/- cells do not migrate in response to low levels of TNF. Higher concentrations of TNF block migration in this cell line and completely inhibit EGF-stimulated MCER2-/- migration (Fig. 3B). However, low levels of TNF stimulate migration in MCER2-/- cells transfected with vector containing full-length TNFR2 but not empty vector (Fig. 3C). Interestingly, both MCER2-/- and MCER1-/- cells migrate more slowly than either YAMC or MSIE cells. Therefore, data from these null colon cells are shown at 24 h after wounding. These findings support a model in which TNFR2 mediates TNF-stimulated intestinal epithelial wound closure and ligation of TNFR1 inhibits growth factor-mediated cell migration.


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Fig. 3.   TNFR2 promotes intestinal cell wound closure. MSIE cells were prepared and wounded as in Fig. 2. Migration was studied in the presence of TNF or EGF with antagonist antibodies to TNFR1 or TNFR2 as indicated (A). The relative change 8 h after wounding is shown. TNFR2-/- mouse colon epithelial (MCER2-/-) cells (B) or cells transfected with full-length TNFR2 (pRK7-p75) or vector (pRK7) (C) were wounded and then cultured with TNF or EGF. The relative change 24 h after wounding is shown. Bars indicate means ± SD. *P < 0.0001 vs. control; @P < 0.0001 vs. EGF treatment; psi  P < 0.01 vs. TNF (1 ng/ml).

Stimulation of cell migration by TNFR2 is associated with catalytic activities of FAK and Src. We (42) and others (38) have previously shown a requirement for tyrosine kinase activity during the process of cellular migration. However, TNFR2 does not contain an intrinsic tyrosine kinase (52). The cytoplasmic tyrosine kinase Src is important for migration of a number of cell types (21, 38, 55). To determine whether TNFR2-mediated migration requires Src tyrosine kinase activity, we studied YAMC cells in the presence or absence of the Src tyrosine kinase-specific inhibitors PD-161430, PP1, or PP2. As shown in Fig. 4, the Src inhibitors block the ability of low-dose TNF to stimulate migration. Molecular dissection of focal adhesion tyrosine phosphorylation sites reveals tyrosine phosphorylation increases on nonreceptor protein tyrosine kinase FAK residue 576 during cell adhesion to fibronectin (14, 15). FAK Y576 is a substrate of activated Src kinase (15). To investigate the effect of TNFR2 stimulation on FAK Y576 phosphorylation, we added low-dose TNF (1 ng/ml) to YAMC cell monolayers in the presence of antagonist antibodies to either TNFR2 or TNFR1. Low-dose TNF induces tyrosine phosphorylation of FAK on residue 576 (Fig. 5A). Blocking TNFR2 inhibits this effect; however, TNFR1 does not alter FAK Y576 phosphorylation. Isolated TNFR2 activation with an agonist antibody is sufficient to induce this increased phosphorylation (Fig. 5A). We further studied the effect of the Src tyrosine kinase-specific inhibitor on TNF-induced phosphorylation on FAK Y576. Preincubation of cells with the Src inhibitor reduces TNF-stimulated FAK Y576 phosphorylation (Fig. 5B).


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Fig. 4.   An inhibitor of Src tyrosine kinase blocks TNF-stimulated migration. YAMC cells were prepared and wounded as in Fig. 1. Cells were treated with EGF (10 ng/ml) or TNF (1 ng/ml) in the presence or absence of Src tyrosine kinase inhibitors PD-161430 (1 µM), PP1 (1 µM), or PP2 (1 µM). The relative change 8 h after wounding is shown. Bars indicate means ± SD. *P < 0.001 vs. control; @P < 0.001 vs. EGF treatment; psi P < 0.001 vs. TNF (1 ng/ml).



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Fig. 5.   TNFR2 regulates focal adhesion kinase (FAK) Y576 phosphorylation. YAMC cells were treated with TNF (1 ng/ml, 15 min) in the presence of an antagonist antibody to TNFR2 or TNFR1, with only an agonist activating TNFR2 (A) or in the presence or absence of Src inhibitor PD-161430 or DMSO (B). Cellular lysates were prepared for Western blot analysis with an antibody against FAK tyrosine phosphorylated on residue 576 (FAK-PY576).

Because Src activation appears necessary for both FAK Y576 phosphorylation and TNF-stimulated intestinal cell migration, we asked whether TNFR2 was required for Src activation. YAMC and MCER2-/- cells were treated with TNF or EGF, and we studied activation by Western blot analysis to determine Src phosphorylation on tyrosine 416 in the Src activation loop (13, 16). Both low and high concentrations of TNF increase Src Y416 phosphorylation in YAMC cells (Fig. 6A). However, Src activation by low-dose TNF treatment is blocked in MCER2-/- but not MCER1-/- cells (Fig. 6A). Loss of TNFR1 has no effect on TNF-stimulated Src activation at either concentration, and EGF stimulates Src activation in the absence of either TNF receptor (Fig. 6A). Furthermore, expressing the intact full-length TNFR2 (pRK7-p75) in these cells restores Src activation by low-dose TNF (Fig. 6B). Thus TNFR2-initiated signal transduction pathways stimulate Src tyrosine kinase activation and FAK tyrosine phosphorylation state and regulate intestinal epithelial cell migration. However, Src activation alone is not sufficient to stimulate cell migration, because higher TNF levels activate Src through TNFR1 yet in the absence of TNFR2 the migration rate is not enhanced.


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Fig. 6.   Low-dose TNF stimulates Src activation through TNFR2. YAMC, MCER1-/-, and MCER2-/- cells (A) and MCER2-/- cells transfected with pRK7-p75 or pRK7 (B), as in Fig. 3, were treated with EGF for 5 min and TNF for 15 min at the indicated concentrations. Cellular lysates were prepared for Western blot analysis with a phospho-Src-tyrosine 416 antibody that detects activated Src.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have shown that TNFR differentially regulate intestinal epithelial cell migration by studying a wound closure assay with three different approaches. First, we observed a concentration-dependent effect with low TNF concentrations enhancing migration and higher concentrations inhibiting migration. An immunoregulatory approach using agonist and antagonist antibodies shows that TNFR2 promotes cellular migration and TNFR1 inhibits this response. Second, loss of TNFR2 results in intestinal cells that no longer migrate in response to TNF. Furthermore, the loss of TNFR1 prevents the antimigratory effect of high TNF levels. Third, loss of TNFR2 results in inhibition of TNF-stimulated Src activation. Src is a necessary kinase for TNF-induced migration and is restored by forced TNFR2 expression. These data suggest that cell migration may be regulated by local TNF levels, with implications for altered wound healing in IBD.

Studies in cell culture and in vivo suggest that the two TNF receptors mediate distinct biological responses from proliferation to inflammation, bone resorption, and vascular permeability (1, 11, 22, 39, 50). Because TNF is pathogenic in IBD, we initiated studies to address the role of TNFR in an in vitro epithelial cell wound closure model. We found that ligation of TNFR1 or TNFR2 mediates opposing effects on wound closure. The development of receptor-deficient mice underlines the potential importance of understanding the individual receptor role in mediating TNF responses. For example, increasing TNF levels by deleting TNF AU-rich elements (Delta ARE) is sufficient to induce IBD in a mouse (24). However, no intestinal inflammation occurred with expression of Delta ARE in TNFR1-/- mice. When expressed on the TNFR2-/- background there was attenuation of gastrointestinal inflammation. Differential regulation of cellular migration by TNFR has been shown to occur in nonepithelial cells of the epidermis as well. TNF promotes migration of cutaneous dendritic cells through the selective activation of TNFR2 (47). Whereas the migration of epidermal Langerhans cells is markedly depressed in p75-deficient mice, it is normal in p55-deficient mice (53). Likewise, in hematopoietic cells TNF enhances the chemotaxis of B cells through TNFR2 (8).

Through development of intestinal epithelial cells expressing only one of the two receptors, we are clearly able to assign distinct biological function to each receptor. In similar studies Abu-Amer and colleagues (1) showed that TNFR1 enhances osteoclastogenesis whereas TNFR2 suppresses this response. Our findings in this report further support a role for low levels of TNF as beneficial to intestinal epithelial integrity, because TNFR2 also stimulates proliferation (22). Although the intracellular events that control cell migration are highly complex and remain incompletely understood, a number of intracellular molecules have been implicated in regulating the initial response to cell injury and/or the resultant migration (57). Interestingly, in cells lacking TNFR1 we find reduced migratory responsiveness by endogenous TNFR2 (Fig. 2B), suggesting additional mechanisms of regulation. One possibility is the induction of TNFR2 shedding reported with high levels of TNF (44). Rapid shedding of TNFR2 consequently reduces cell sensitivity to TNF (2), supporting a role for endogenously produced soluble p75 in downregulating TNF-driven responses (39). Another possibility is the so-called ligand passing hypothesis (49). This model suggests enhanced receptor responsiveness of TNFR1 by ligand passing from TNFR2 and therefore seems less likely to explain our observation than does receptor shedding.

Tyrosine kinase activity is necessary for intestinal epithelial cell migration (5, 6, 38, 41, 46). Therefore, in the absence of any endogenous TNFR2 catalytic activity (52) we speculated that intracellular nonreceptor protein kinases mediate TNF-stimulated cell migration. A screen for intermediates involved in signal transduction pathways suggested Src family members as candidate tyrosine kinases regulating TNF-stimulated migration. These cytoplasmic tyrosine kinases participate in a wide variety of cellular responses, including adhesion and motility (38). We found that inhibiting Src tyrosine kinase decreases TNF-induced FAK tyrosine 576 phosphorylation, a Src phosphorylation site on FAK (15). This finding suggests a potential role for Src kinase in low-dose TNF-stimulated migration through activation of FAK. Clearly, FAK-null cells have reduced motility (20). Furthermore, the ability of FAK to regulate migration is dependent on site-specific tyrosine phosphorylation including tyrosine 576 (37). It is likely that phosphorylation on these sites determines FAK interaction with several intracellular signaling molecules including Src family kinases (26). Given this background the data reported here suggest that TNFR2 activates a novel Src-regulated pathway enhancing migration of intestinal epithelial cells involving FAK tyrosine phosphorylation.

The role of Src activation by high TNF levels through TNFR1 in reducing cell migration rate remains unclear. Expression of dominant-negative Src in YAMC cells alters cellular morphology, and the pharmacological inhibition of Src kinase activity in the presence of high TNF induced near complete apoptosis of YAMC cells (data not shown). Although little information is available regarding Src as an antiapoptotic molecule, loss of Src kinase activity enhances osteoclast apoptosis through TNFR1 (58). Furthermore, the loss of Src activity in the acid sphingomyelinase-null mouse enhances TNF family member Fas-induced apoptosis (35). Therefore, the activation of Src via TNFR1 may represent a novel antiapoptotic pathway in intestinal epithelial cells. Clearly, this observation warrants further study.

In conclusion, we have used several approaches to examine the migration of intestinal epithelial cells to a range of TNF levels. We report that TNFR2 stimulates intestinal cell migration, whereas TNFR1 inhibits migration in an in vitro wound closure assay. Receptor-dependent TNF-regulated responses have been reported in other tissues; however, the novel observations of these studies are the TNFR2-stimulated activation of Src and the direct regulation of epithelial cell migration by TNF. The potential roles for these receptors in regulating epithelial monolayer homeostasis in an environment of high TNF levels warrant further investigation with implications for mucosal healing and IBD.


    ACKNOWLEDGEMENTS

We thank Peter Dempsey and Steve Hanks for helpful discussions and for critically reviewing the manuscript.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-56008 (D. B. Polk) and DK-58404 (the Vanderbilt University Digestive Disease Research Center).

Address for reprint requests and other correspondence: D. B. Polk, Dept. of Pediatrics, Division of Gastroenterology, Hepatology and Nutrition, 21st and Garland Ave., S-4322 MCN, Nashville, TN 37232-2576 (E-mail: d-brent.polk{at}vanderbilt.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published December 4, 2002;10.1152/ajpcell.00309.2002

Received 2 July 2002; accepted in final form 27 November 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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