Activation of the RON receptor tyrosine kinase attenuates transforming growth factor-ß1-induced apoptotic death and promotes phenotypic changes in mouse intestinal epithelial cells
Da Wang1,2,
Qi Shen2,
Xiang-Ming Xu2,
Yi-Qing Chen2 and
Ming-Hai Wang1,3
1 Laboratory of Cheung-Kong Scholars Program for Biomedical Sciences, Institute of Infectious Diseases, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, P. R. China and 2 Department of Pharmaceutical Sciences and Center for Cancer Biology, Texas Tech University Health Sciences Center School of Pharmacy, Amarillo, TX 79106, USA
3 To whom correspondence should be addressed Email: minghai.wang{at}ttuhsc.edu
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Abstract
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The RON (recepteur d'origine nantais) receptor belongs to the MET proto-oncogene family that is implicated in the oncogenesis of the gastrointestinal epithelium. The present study aimed to determine the role of RON in regulating epithelial phenotypes in response to transforming growth factor (TGF)-ß1. Expression and activation of RON in SV40-immortalized mouse intestinal epithelial MODE-K cells result in reduction of cellular sensitivities towards apoptotic signals elicited by TGF-ß1. This effect is dependent on RON expression and phosphorylation that inhibit the TGF-ß1-induced activation of caspase-3 and truncation of BAD. Among cellular signaling components, the activation of MAP kinase is critical in the RON-mediated inhibitory effect. PD98059, a specific MAP kinase inhibitor, prevented RON-mediated anti-apoptotic activities. PD98059 also prevented the inhibitory effect of RON on TGF-ß1-induced cleavage of caspase-3 and BAD. By protecting cells from apoptotic death, activated RON collaborates with TGF-ß1 in the induction of cell morphological changes with decreased E-cadherin expression and increased migration and morphogenesis. Thus, RON expression and activation modulate phenotypes of gastrointestinal epithelial cells in response to TGF-ß1 with reduced sensitivity to apoptosis and increased migration. These activities might represent a mechanism by which RON activation increases tumorigenic activities and facilitates the progression of transformed epithelial cells towards malignancy.
Abbreviations: mAb, monoclonal antibody; DMEM, Dulbecco's modified Eagle's medium; EMT, epithelial to mesenchymal transition; FBS, fetal bovine serum; MSP, macrophage stimulating protein; RON, recepteur d'origine nantais; si, small interfering; TGF, transforming-growth factor
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Introduction
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Gastrointestinal epithelium maintains a delicate balance based on continuous coordination between growth and apoptosis (1). This coordination is regulated precisely through interaction with and response to various extracellular signals (2,3). A variety of growth factors and cytokines, such as hepatocyte growth factor (HGF)/scatter factor and transforming growth factor (TGF)-ß1, are involved in this process (2,3). These growth factors, by binding to their specific cell surface receptors, form regulatory autocrineparacrine loops that control epithelial cell replication and apoptotic death (2,3).
During cellular transformation and tumor progression, certain gastrointestinal epithelial cells gradually lose their sensitivities towards extracellular balanced signals (4,5). This leads to reduced apoptotic death and increased cell proliferation. The responsiveness of intestinal epithelial cells to TGF-ß1 is an example (5,6). Normal or immortalized intestinal epithelial cells undergo apoptotic death upon interaction with TGF-ß1 (57). In contrast, tumorigenic and malignant cells respond to TGF-ß1 with increased tumorigenic activities leading to malignant phenotypes (57). Thus, the insensitivity of intestinal epithelial cells towards TGF-ß1 is an important sign indicating oncogenic transformation with phenotypic alterations. It also suggests that cellular signaling cascades in cancerous cells are disorganized, which allows cells to escape TGF-ß1-induced apoptosis but utilize its oncogenic-promoting activities (57). Currently, the mechanisms underlying the resistance of cancerous cells towards TGF-ß1-induced apoptotic death are still largely unknown, although aberrant TGF-ß1 signaling might be one of the causes (810). Since activation of receptor tyrosine kinases often results in uncontrolled proliferation with reduced apoptotic death, which leads to increased tumorigenic behaviors (11), it is believed that receptor tyrosine kinases have the ability to modulate cellular phenotypes in response to TGF-ß1-induced signals.
The RON (recepteur d'origine nantais) receptor tyrosine kinase is a member of the MET proto-oncogene family (12,13). The ligand for RON has been identified as macrophage stimulating protein (MSP) (1417), also known as HGF-like protein (18). Activation of RON by MSP stimulates multiple signaling cascades including Ras (19), phosphatidylinositol (PI)-3 kinase (20), MAP kinase (21) and NF
kB (22). These pathways control a variety of cellular activities in different types of cells (1922). Accumulated evidence has showed that altered RON expression is involved in the progression of certain gastrointestinal epithelial tumors including those from the colon (23,24). Increased proliferation with reduced sensitivity to apoptotic signals in normal human colon epithelial cells expressing RON has been documented (23). However, the mechanisms by which RON regulates phenotypic changes in gastrointestinal epithelial cells are unknown.
The present work studied whether RON activation regulates epithelial cells in response to TGF-ß1 signals leading to increased tumorigenic activities. Our results showed that RON activation protects immortalized intestinal epithelial cells from TGF-ß1-induced apoptosis and enhances cellular tumorigenic activities. These activities were mediated by RON-transduced signals that activate the mitogen-activated protein kinase (MAPK)/ERK pathway. Our results also showed that activated RON collaborated with TGF-ß1 in the induction of epithelial morphological changes with reduced E-cadherin expression. Thus, increased RON expression and activation have a profound impact on gastrointestinal epithelial cells that might contribute to their oncogenic process in vivo.
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Materials and methods
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Cells and reagents
SV40 large T antigen-immortalized mouse intestinal epithelial MODE-K cells (25) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 10 µg/ml of insulin. Human MSP was provided by Dr E.J.Leonard (National Cancer Institute, Frederick, MD). Human TGF-ß1 was from R&D Systems (Minneapolis, MN). Mouse monoclonal antibody (mAb) (clone ID2) and rabbit IgG antibodies to RON were used as described previously (26). Mouse mAb to pan-ERK were from Transduction Laboratories (San Diego, CA). Mouse mAb to p-ERK, phospho-tyrosine, E-cadherin or rabbit antibodies to caspase-3 were from Cell Signaling (Beverly, MA). Mouse mAb to BAD and goat IgG to actin were from Santa Cruz Biotechnology (Santa Cruz, CA). PD98059, SB203580 and Wortmannin were from Calbiochem (San Diego, CA).
Establishment of MODE-K cell lines expressing RON and its mutant m1254t
The cDNA encoding human RON or RON mutant m1254t was inserted into the pTracer-CMV/BSD vector (Invitrogen). Transfected cells were selected in 3 µg/ml of blasticidin. Cells expressing RON (MODE-RON) or RONm1254t (MODE-RONmt) were isolated by positive selection with Dynal beads (Dynal) coated with mAb ID2. RON expression was determined by western blotting using rabbit IgG to RON (23).
Immunoprecipitation and western blot analysis
The methods were performed as detailed previously (14). The mAb ID2 was used for RON immunoprecipitation. Rabbit IgG to RON or other antibodies were used in western blotting. The reaction was developed with enhanced chemiluminescence reagents and recorded on film.
Cell apoptotic assays
Parental or transfected MODE-K cells (1 x 106 cells/dish) in DMEM with 2% FBS were pre-treated with 5 nM of MSP and then stimulated with 2 ng/ml of TGF-ß1 or 1 µg/ml of Fas-activating antibody. Cell viabilities were determined by the Hoechst day 3258 assay 24 h after treatment as described previously (27). A minimum of 200 cells was counted in each sample. In some experiments, the TUNEL assay (DeadEnd colorimetric kit, Promega, Madison, MI) was also used to validate the results.
Generation of RON mRNA silencing vectors and cell transfection
The small interfering (si)RNA sequences were designed using standard selection rules (28). A 55-nt that encodes two complementary sequences of 21 nt, corresponding to the RON coding sequence in exon 1 (29), separated by a hairpin structure, was inserted into psiRNA-hH1zeo (Invivogen, San Diego, CA), yielding the vector RON-specific siRNA (psiR-RON). A control vector psiRmRON containing the similar RON sequence with four mutations was also generated. Transfection of MODE-RON cells with psiR-RON vector was performed as described (24). The effect of siRNA on silencing RON expression was determined by western blot analysis.
Cell migration and morphogenesis assays
Migration of MODE-RON cells into an open area on the surface of a culture dish was determined by using a previously described method (30). Briefly, cells were stimulated with MSP, TGF-ß1, or both for 24 h, fixed, and then photographed. Cell morphogenic activities were determined by using the collagen gel assay as described previously (31). Morphogenic activities were determined 7 days after initiation of cell culture and photographed.
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Results
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Expression of RON and RONm1254t in mouse intestinal epithelial cells
MODE-K cells were from the small intestine, immortalized by SV40 large T antigen (25), and did not express mouse RON as evident in RTPCR and western blot analysis (data not shown). Cells maintained epithelial phenotypes and were non-tumorigenic in vivo (25). Results in Figure 1 showed levels of RON or RONm1254t expressed in stably transfected cells (designated as MODE-RON and MODE-RONmt cells, respectively). The presence of mature RON was evident by detecting the RON-ß chain (Figure 1A). As expected, RON was not detected in parental MODE-K cells. Results from the tyrosine phosphorylation assay showed that MSP induces RON phosphorylation in MODE-RON cells. RONmt was constitutively active. MSP stimulation further increased its tyrosine phosphorylation (Figure 1B).

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Fig. 1. Expression and phosphorylation of RON or RONm1254t inMODE-K cells: MODE-K, MODE-RON or MODE-RONmt cells (3 x 106 cells/culture dish) were stimulated with or without 5 nM of MSP for 10 min at 37°C. Immunoprecipitation was performed using cell lysates with mAb ID2 (1 µg) bound to protein GSepharose beads. Proteins were separated in a 7.5% SDSPAGE under reduced conditions and transferred to the membrane. RON or RONmt was detected by rabbit IgG antibodies to RON (A). Phosphorylated RON or RONmt was detected by anti-P-tyr-100 (PY) antibodies (B).
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Effect of MSP on TGF-ß1-induced apoptotic death of MODE-K cells
To determine if RON expression changes cellular sensitivities towards apoptotic signals, MODE-RON cells were pre-treated with 5 nM of MSP and then stimulated with 2 ng/ml of TGF-ß1. Cells stimulated with Fas-activating antibodies were used as the positive control. The percentage of apoptotic cells was determined by Hoechst dye 33258 nuclear staining. Results are shown in Figure 2. After exposing MODE-RON cells to TGF-ß1, the percentage of dead cells was dramatically increased from baseline (
5%) to 29%, which was comparable with those induced by Fas-activating antibodies (28%). MSP alone had no effect on cell viability; however, when cells were stimulated with TGF-ß1 in the presence of MSP, the percentage of apoptotic cells was reduced to 10%. In contrast, TGF-ß1-induced apoptosis of parental MODE-K cells was not inhibited by MSP. Results from TUNEL assays also confirmed the inhibitory effect of MSP on TGF-ß1-induced apoptosis. The percentage of apoptotic cells induced by TGF-ß1 (31.7%) was reduced to 10.8% when MSP was added to cell cultures. These results indicate that MSP-induced RON activation protects MODE-K cells against TGF-ß1-induced apoptotic death.

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Fig. 2. Effect of RON activation on TGF-ß1-induced apoptotic death of MODE-RON cells: parental MODE-K or MODE-RON cells (5 x 105 cells/dish) were cultured overnight in DMEM with 10% FBS and then switched to DMEM with 2% FBS. Cells were pre-treated with 5 nM of MSP for 30 min and then stimulated with or without 2 ng/ml of TGF-ß1 for 24 h. Cells treated with Fas-activating antibodies (FasAb) were used as the positive control. Apoptotic cells were determined by Hoechst dye 33258 nuclear staining (27). The percentage of apoptotic cells was calculated from total numbers of cells. Spontaneously apoptotic death was at 5%. Results shown here are from one of two experiments.
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Kinetic- and dose-dependent activities of MSP in TGF-ß1-induced apoptotic death
The time-course of MSP-induced inhibition is shown in Figure 3. As presented in Figure 3A, low levels of spontaneous apoptosis occurred when the cells were incubated in the culture medium. Upon TGF-ß1 stimulation, the numbers of apoptotic cells increased progressively up to 50% by the end of incubation period (72 h). MSP alone had no effect on cell viabilities; however, when MSP was used together with TGF-ß1, apoptosis was reduced in a time-dependent fashion with only a 20% cell death.

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Fig. 3. Kinetic and concentration-dependent effects of MSP on TGF-ß1-induced apoptosis. MODE-RON cells (5 x 105 cells/dish) were cultured as described in Figure 2. (A) MODE-RON cells were simultaneously treated with 2 ng/ml of TGF-ß1, 5 nM of MSP, or both and the percentage of apoptotic cells was determined at different time intervals. (B) Cells were pre-treated with different amounts of MSP for 30 min and then stimulated with 2 ng/ml of TGF-ß1 for 24 h. The percentage of apoptotic cells in (A and B) was determined by Hoechst dye 33258 staining. Results shown here are from one of two experiments with similar results.
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We further tested if the MSP-induced inhibition is dependent on the concentration of MSP. The percentage of apoptotic cells was determined after cells were incubated for 48 h. Results are shown in Figure 3B. MSP inhibited TGF-ß1-induced apoptosis in a concentration-dependent manner. Maximal inhibition was observed with 10 nM of MSP. A further increase in the amount of MSP did not lead to an additional inhibition (data not shown).
Requirement of RON expression and activation in inhibiting TGF-ß1-induced apoptosis
To study MSP-induced anti-apoptotic activities in more detail, we first determined if RON expression is necessary in protecting cells from apoptosis. The siRNA-based RNA interference technique (28) was used. Expression of psiR-RON caused a significant reduction in RON expression (Figure 4A). The RON ß-chain was reduced to a greater degree than that of the RON precursor, which might reflect the reduced conversion of the RON precursor in psiR-RON-treated cells. No silencing effect was seen when the control vector psiRmRON was used. Since MODE-K cells do not express mouse RON as determined by specific RTPCR and western blot analysis (data not shown), the influence of the endogenous receptor can be excluded. We also tested the activities of siRNA on the expression of mouse MET, a protein with similar structures to RON. Results from western blot analysis showed that RON-specific siRNA does not affect MET expression (data not shown), indicating that the siRNA is specific to RON.

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Fig. 4. Effect of silencing RON expression on TGF-ß1-induced apoptosis: MODE-RON cells (1 x 106 cells/dish) were transiently transfected with the expression vector psiR-RON or control vector psiRmRON for 48 h. (A) Western blot analysis of RON expression by MODE-RON cells transfected with psiR-RON. (B) Effect of psiR-RON on TGF-ß1-induced apoptosis of MODE-RON cells transfected with psiR-RON. Apoptotic cells were identified by Hoechst dye 33258 staining.
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Transfection of MODE-RON cells with psiR-RON or psiRmRON did not result in any change in the level of spontaneous or TGF-ß1-induced apoptosis (Figure 4B). By silencing RON expression, the number of apoptotic cells induced by TGF-ß1 was increased in the presence of MSP to 25% of apoptotic cells. The partially recovered apoptotic effect of TGF-ß1 is probably due to transfected cells expressing psiR-RON. It is estimated by using a psi-vector expressing the GFP protein that only 60% cells were transfected by psiR-RON. The effect of psiRmRON on TGF-ß1-induced apoptosis was not observed. The number of cells undergoing apoptotic death was still inhibited by MSP. These results demonstrate that RON expression and activation are required for MSP to inhibit TGF-ß1-induced apoptotic death.
MODE-RONmt cells were also used to determine if RON activation is required for MSP-induced inhibition. As shown in Figure 5A, the apoptotic effect of TGF-ß1 on MODE-RONmt cells was minimal as indicated by Hoechst dye 33258 staining. Only 5% apoptosis, similar to spontaneous apoptosis, was documented (Figure 5B). Thus, RON expression and activation are important in the protection of MODE-K cells against TGF-ß1-induced apoptosis.

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Fig. 5. Requirement of RON activation in inhibition of TGF-ß1-induced apoptosis: MODE-RON or MODE-RONmt cells (5 x 105 cells/dish) were stimulated with MSP, TGF-ß1, or both as described in Figure 2. (A) Reduced apoptotic death in MODE-RONmt cells as determined by Hoechst dye 33258 staining. (B) Comparison of TGF-ß1-induced apoptosis between MODE-RON and MODE-RONmt cells as determined by Hoechst dye 33258 staining. Results shown here are from one of three experiments with similar results.
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Effect of RON activation on TGF-ß1-induced activation of caspase-3 and BAD
To determine if MSP-induced inhibition is mediated by regulating the activities of pro-apoptotic proteins, the effect of RON activation on TGF-ß1-induced activation of caspase-3 and BAD was studied. Treatment of MODE-RON cells with TGF-ß1 resulted in the activation of caspase 3, which was evident by the presence of a 17-kDa cleaved fragment of caspase-3 (Figure 6A). MSP alone had no effect on caspase-3 activation; however, when MSP was used with TGF-ß1, the TGF-ß1-induced cleavage of caspase-3 was inhibited.

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Fig. 6. Inhibitory effect of MSP on TGF-ß1-induced activation of caspase 3 and BAD: experiments were performed as described in Figure 2. MODE-RON cells were used for stimulation. Western blot analysis was carried out as detailed in the Materials and methods. The effect of MSP on TGF-ß1-induced activation of caspase-3 was shown in (A). The cleaved caspase fragment was indicated by the arrow. The effect of MSP on TGF-ß1-induced cleavage of Bad was shown in (B). The fragment was indicated by the arrow. The effect of MSP on Bcl-XL expression was shown in (C). The same membrane was treated with erase buffer and re-probed with antibodies to ß-actin as the loading control (D). One of three experiments with similar results.
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We also observed that RON activation inhibits TGF-ß1-induced cleavage of BAD. As shown in Figure 6B, BAD was truncated with the appearance of a 15-kDa protein when MODE-RON cells were treated with TGF-ß1. Upon MSP treatment, the truncated 15-kDa protein from BAD was diminished, indicating that the truncation of BAD is inhibited. No effect was observed on Bcl-XL expression (Figure 6C). Thus, results from Figure 6 demonstrate that RON activation inhibits TGF-ß1-induced activation of caspase-3 and BAD.
Preventive effect of PD98059 on RON-mediated inhibition of apoptotic activities-induced by TGF-ß1
The above results prompted us to study possible signaling components involved in RON-mediated anti-apoptotic activities. MAPK/ERK was chosen because of its involvement in protecting cells from apoptosis (32,33). We first determined MSP-induced ERK1/2 phosphorylation in western blot analysis. As shown in Figure 7A, ERK1/2 phosphorylation was hardly detected in MODE-RON cells without MSP stimulation. After MSP stimulation, levels of ERK1/2 phosphorylation increased rapidly and reached its peak at
3060 min and then gradually reduced. In MODE-RONmt cells that were serum-starved overnight, low levels of spontaneous ERK1/2 phosphorylation were observed in unstimulated cells (Figure 7B). Increased phosphorylation was seen upon MSP stimulation. The peak of phosphorylation reached at 15 min and gradually reduced to the baseline at 120 min (Figure 7B). As shown in Figure 7C, TGF-ß1 alone had no effect on ERK1/2 phosphorylation. It also had no effect on RON-mediated ERK1/2 phosphorylation. To determine if PD98059, a specific MAP kinase inhibitor, regulates RON-mediated ERK1/2 phosphorylation, cells were pre-treated with PD98059 and then stimulated with MSP. As shown in Figure 7D, PD98059 completely inhibited ERK1/2 phosphorylation induced by MSP in both MODE-RON and MODE-RONmt cells.

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Fig. 7. RON-mediated ERK1/2 phosphorylation and its regulation by TGF-ß1 or PD98059: MODE-RON (A) and MODE-RONmt (B) cells (2 x 106 cells/dish) were serum-starved overnight and then stimulated with 5 nM of MSP at different time intervals. MODE-RON cells were also stimulated for 30 min with MSP, 2 ng/ml of TGF-ß1, or both (C). To determine the effect of PD98059, MODE-RON or MODE-RONmt cells were stimulated with MSP for 30 min in the presence of 40 µM of PD98059 (D). In all experiments, cells without stimulation were used as the control. Phosphorylated ERK1/2 was detected by western blotting using antibodies to phosphor-p42 and p44. The same membrane was also re-probed with the antibodies to p42. Actin was used as the loading control. One of the two experiments with similar results.
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To determine if PD98059 prevents RON-mediated inhibition of apoptosis, MODE-RON or MODE-RONmt cells were pre-treated with 40 µM of PD98059 and then stimulated with TGF-ß1 in the presence or absence of MSP. As shown in Figure 8A, RON activation inhibited TGF-ß1-induced apoptotic death of MODE-RON cells. The inhibition was prevented when PD98059 was used. The preventive effect of PD98059 was also confirmed when MODE-RONmt cells were used (Figure 8B). Expression of RONm1254t protected cells from TGF-ß1-induced apoptotic death. These activities were diminished when PD98059 was present, resulting in a 20% apoptotic death. Interestingly, the preventive effect of PD98059 on RON-mediated inhibition of apoptosis by Fas-activating antibodies was not observed (data not shown), indicating that MSP-induced Erk1/2 activation alone is not sufficient to antagonize apoptotic signals elicited by Fas-activating antibodies. In addition, no significant effect was observed when wortmannin or SB203580 was used (Figure 8A and B). Thus, RON-mediated ERK1/2 activation is important in inhibiting TGF-ß1-induced apoptotic death of MODE-K cells.

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Fig. 8. Preventive activities of PD98059 in MSP-induced inhibition of apoptosis: MODE-RON (A) or MODE-RONmt (B) cells were stimulated in triplicate as described in Figure 2. PD98059 (40 µM) was added in cell cultures with MSP, TGF-ß1, or both. The percentage of apoptotic cells was determined by Hoechst dye 33258 staining after cells were incubated for 24 h. CTL, control; M, MSP; PD, PD98059; WT, Wortmannin; SB, SB203580. Experiments were repeated twice. Results shown here are from one representative experiment.
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Effect of PD98059 on RON-mediated inhibition of caspase-3 and BAD activation
Since RON activation inhibits TGF-ß1-induced activation of caspase-3 and BAD, we wanted to know if PD98059 is involved in these events. To this end, MODE-RON or MODE-RONmt cells were pre-treated with PD98059 and stimulated with TGF-ß1 in the presence or absence of MSP. As shown in Figure 9A, TGF-ß1-activated caspase-3 was inhibited by MSP. Treatment of cells with PD98059 alone did not cause caspase-3 fragmentation (data not shown). When PD98059 was included with TGF-ß1 and MSP, the cleaved 17-kDa caspase-3 fragment reappeared, suggesting that MSP-induced inhibition of caspase-3 activation was prevented. Similar results were also found when MODE-RONmt cells were used, in which the TGF-ß1-induced caspase-3 fragment was detected when PD98059 was included (Figure 9B).

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Fig. 9. Effect of PD98059 on MSP-induced inhibition of caspase-3 and BAD activation: MODE-RON or MODE-RONmt cells were treated as described in Figure 8. Proteins (50 µg/sample) were separated in 12% SDSPAGE followed by western blot analysis using antibodies specific to caspase-3 (A and B) or BAD (C). Membranes were also re-probed with antibodies to ß-actin for the loading control. One of three experiments with similar results.
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The effect of PD98059 on BAD activation is shown in Figure 9C. PD98059 prevented RON-mediated inhibition of the BAD truncation-induced TGF-ß1. Similar results were also observed in MODE-RONmt cells (data not shown). These results indicate that the preventive effect of PD98059 is associated with its ability to restore TGF-ß1-induced activation of caspase-3 and BAD.
Regulation by MSP of MODE-K cell morphologies in the presence of TGF-ß1
To study if RON-mediated protective activities are associated with cell-phenotypic change, the effect of MSP in collaboration with TGF-ß1 on MODE-K cell motilities was determined. As shown in Figure 10A, MODE-RON cells displayed typical epithelial morphologies with tight adherens adjunction. The treatment of cells with MSP caused a marginal morphological effect with a slight increase in cell length as indicated by the elongation index. Although TGF-ß1-induced a massive cell apoptosis, it had a profound morphological effect on the remaining cells with significant cell elongation. As expected, the majority of MODE-RON cells were viable when MSP was used together with TGF-ß1. Interestingly, cell morphologies gradually changed to a fibroblast-like appearance. By measuring cell length, the elongation index in these cells was increased almost 4-fold in comparison with those from control cells.

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Fig. 10. Collaborative activities of MSP and TGF-ß1 in induction of MODE-RON cell morphological changes and E-cadherin expression. (A) MODE-RON cells were stimulated with TGF-ß1 (2 ng/ml), MSP (5 nM), or both for 48 h. Morphological changes were examined under the microscope and photographed (magnification x200). The elongation index (B) was obtained by measuring the length of 200 randomly selected cells from each group. The length of control cells was set as 1.0. The elongation index was calculated by dividing lengths of experimental cells with lengths of control cells. (C) The effect of MSP and TGF-ß1 on E-cadherin expression was determined by western blot analysis using antibodies to E-cadherin. MODE-RON cells were treated as described in (A) and incubated for 7 days. One of two experiments with similar results.
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Since transition of epithelial cells towards fibroblast-like morphologies is often accompanied with reduced epithelial cell properties and increased expression of mesenchymal markers (34), we wanted to determine if the expression of E-cadherin, an epithelial cellular marker, in MODE-RON cells is reduced. As shown in Figure 10B, levels of E-cadherin 7 days after TGF-ß1 stimulation were significantly lower than those in control cells. Although it alone had no effect, MSP further reduced E-cadherin expression in the presence of TGF-ß1. Interestingly, expression of vimentin or
-smooth muscle actin, both mesenchymal cellular markers, was not detected in western blotting (data not shown). Thus, MSP collaborates with TGF-ß1 to induce fibroblast-like morphologies in MODE-RON cells with reduction of E-cadherin, but not mesenchymal marker expression.
Effect of RON activation on MODE-K cell migration/morphogenesis
Migration of MSP-stimulated MODE-RON cells was determined by measuring cells that moved into the open space on the surface of a culture dish. As shown in Figure 11A, MSP or TGF-ß1 alone induced cell migration. More than 50% of the open space was covered within 24 h by migrated cells. This effect was further enhanced when MSP was used together with TGF-ß1. Almost all of the open space was occupied with migrated cells stimulated with MSP and TGF-ß1.

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Fig. 11. Increased motile and morphogenic activities of MODE-RON cells in response to MSP and TGF-ß1. (A) Migration of MODE-RON cells. Experiments were performed as described previously (29). After stimulation of cells for 24 h, cells were fixed and photographed (magnification x100). (B) Morphogenesis of MODE-RON cells in collagen gel. Cells cultured in collagen gel were stimulated with MSP (5 nM), TGF-ß1 (2 ng/ml), or both for 7 days and then photographed. Results shown here are from one of two experiments with similar results.
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To determine if RON activation results in tubulogenesis, MODE-RON cells were cultured in collagen gel and stimulated with MSP, TGF-ß1, or both. As shown in Figure 11B, control or TGF-ß1-stimulated cells did not form cell cords that invade collagen gel. In contrast, MSP induced tubulogenesis in MODE-RON cells with the formation of medium-sized branches that infiltrated into collagen gels. In cells stimulated with MSP and TGF-ß1, tubulogenic activities were further increased. Numerous cell branches were formed, which penetrated deeply into the collagen gel. Also, cell branches were much longer than those induced by MSP alone. These results, together with those in Figure 11A, demonstrate that RON activation, in collaboration with TGF-ß1, is capable of inducing motile and morphogenic phenotypes in MODE-RON cells.
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Discussion
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The progression of gastrointestinal epithelial cells towards the tumorigenic phenotype is a complicated process characterized by an accumulation of genetic alterations and cellular disorganization (35). The phenotypic change by epithelial cells in response to TGF-ß1 is one of the critical events that determine malignant behaviors (36). Using intestinal MODE-K epithelial cells as the model, we demonstrated that increased RON expression and activation contribute to the acquisition of oncogenic phenotypes by gastrointestinal epithelial cells. We showed that RON expression and activation render MODE-K cells resistant to apoptotic signals elicited by TGF-ß1. These effects were mediated by RON-transduced signals that are sensitive to specific inhibitors of MAP kinase. The conversion of cellular phenotypes was also evident in which MSP collaborates with TGF-ß1 in inducting morphological changes with decreased E-cadherin expression and increased motile activities. Such changes partially resemble epithelial to mesenchymal transition (EMT) (34,37), a process essential for tumor invasion and metastasis. Thus, increased RON expression and activation play an important role in modulating epithelial cell phenotypes in response to TGF-ß1. These effects might have pathological implications during the progression of gastrointestinal epithelial cells towards tumorigenesis.
The evidence that MSP acts as a survival factor for epithelial cells has been reported previously (23,38). We have shown previously that MSP-dependent RON activation protects immortalized human colon epithelial CoTr cells against apoptotic death induced by Fas-activating antibodies (23). Another report demonstrated that MSP protects MDCK cells and keratinocytes from apoptotic death induced by preventing cell adherence (anoikis) or by serum starvation (38). The present results provide additional evidence showing that RON is capable of protecting mouse intestinal epithelial cells from apoptotic death induced by TGF-ß1. As shown in Figures 2

6, RON expression and activation resulted in resistance of MODE-K cells to TGF-ß1-induced apoptosis. The reduced cellular sensitivities towards apoptosis were probably channelled through blocking the TGF-ß1-induced activation of caspase-3 and BAD. These findings are important with regard to RON in regulating cellular sensitivities towards extracellular apoptotic signals. First, activated RON has the ability to protect different types of epithelial cells from extracellular apoptotic signals elicited by TGF-ß1, Fas-activating agents, or other mechanisms, suggesting that RON-transduced survival signals are capable of antagonizing apoptosis elicited through different pathways. Secondly, activated RON transduces signals that render epithelial cells a non-responsive status, which could give transformed cells a chance to grow under unfavorable conditions. Thus, altered RON expression has a profound impact on responsiveness of cells towards extracellular signals. It is possible that RON-mediated survival of epithelial cells is one of the mechanisms that protect transformed cells from elimination under hostile environment.
The differential responsiveness of epithelial cells towards TGF-ß1 is intriguing. TGF-ß1 induces apoptosis in normal epithelial cells but promotes tumorigenic progression in cancerous cells (57). At present, the mechanisms underlying the dual actions of TGF-ß1 are still largely unknown. Disruption of TGF-ß1Smad signaling cascades by somatic mutation in cancer cells is one mechanism (810). However, MODE-K cells are immortalized cells that are sensitive to TGF-ß1-induced apoptotic signals. The deregulated TGF-ß1Smad pathway caused by mutations does not apply to RON-mediated insensitivities towards apoptosis. A potential mechanism for the action of RON is probably channelled by RON-mediated activation of the RasMAP kinase pathways, which are known to sustain cell survival (19,31,33). In RON-activated cells, increased RAS and MAP kinase activities are often observed (19,21,39). We showed that RON activation causes ERK1/2 phosphorylation in a time-dependent manner. It was reported previously that expression of RONm1254t in NIH3T3 cells causes high levels of ERK1/2 activation (39); however, only low levels of spontaneous ERK1/2 phosphorylation were observed in MODE-RONmt cells. We reasoned that the difference is probably related to our experimental conditions, in which cells were serum-starved for a long period. The MSP-dependent or -independent ERK1/2 phosphorylation was inhibited when PD98059, the specific inhibitor of MAP kinase, was used in the cell culture. Moreover, PD98059 prevented MSP-induced anti-apoptotic activities (Figure 8). As demonstrated in MODE-RONmt cells, the effect of TGF-ß1 on apoptosis was diminished, and further reduced in the presence of MSP (Figure 8B). Treatment of cells with PD98059 significantly restored the apoptotic effect of TGF-ß1, indicating that RON-mediated ERK1/2 activation is involved in MSP-induced anti-apoptotic activity. As demonstrated in Figure 9, the inhibitory effect of RON on TGF-ß1-induced activation of caspase-3 was prevented upon treatment of cells with PD98059. Similarly, PD98059 prevented the inhibitory effect of MSP on TGF-ß1-induced truncation of BAD. Thus, activation of the MAP kinase pathways seems to be critical in RON-mediated anti-apoptotic activities. In supporting this notion, it has been shown that the survival of epithelial cells during serum starvation is mediated by MSP-induced activation of MAP kinase, although other mechanisms are also involved (38). At present, we do not know how RON activation inhibits TGF-ß1-induced cleavage of caspase-3 and BAD. It will be of great interest to elucidate mechanisms underlying these activities in the future.
The findings that activated RON collaborates with TGF-ß1 in inducting morphological changes with decreased E-cadherin expression and increased migration are interesting. Both MSP and TGF-ß1 are known for their morphological effects on epithelial cells (40). In TGF-ß1-stimulated cancer cells, a process known as EMT often occurs (34). EMT is characterized by spindle-shaped (fibroblast-like) morphologies with the loss of epithelial markers such as E-cadherin and the gain of mesenchymal phenotypes (34,37). Most invasive and/or metastatic cancers are featured by partial or complete EMT (34,37). As demonstrated in Figure 10, by reducing the sensitivities of MODE-K cells towards apoptotic signals, activated RON collaborated with TGF-ß1 in inducting fibroblast-like morphologies. The morphological changes were accompanied with reduced E-cadherin expression and increased cell migration. We have shown previously that RON activation is required for complete EMT in kidney epithelial MDCK cells stimulated by TGF-ß1 (21); however, we did not find mesenchymal marker expression in MODE-RON cells even though E-cadherin expression was significantly reduced. This is probably due to individual cell types. Nevertheless, our results indicate that in gastrointestinal epithelial MODE-K cells, RON-mediated signals not only antagonize TGF-ß1-induced apoptotic activities, but also collaborates with TGF-ß1 in regulating cellular morphologies and motilities. Such collaboration could be one of the mechanisms by which RON exerts its pathogenic effect on transformed gastrointestinal epithelial cells.
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Acknowledgments
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This work was performed at Texas Tech University Health Sciences Center. We thank Drs G.Gaudino (Universita di Torino, Novara, Italy) for the RONm1254t cDNA and E.J.Leonard (National Cancer Institute, Frederick, MD) for MSP. This work was supported by NIH Grant R01 CA91980, Amarillo Area Foundation, and the Foundation of Cheung Kong Scholars Program from the Chinese Ministry of Education.
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Received April 15, 2004;
revised September 2, 2004;
accepted September 10, 2004.