Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee 38163
Submitted 8 August 2003 ; accepted in final form 10 October 2003
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ABSTRACT |
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apoptosis; c-Jun NH2-terminal kinase; cytochrome c; caspases; camptothecin
TNF-, an important pleiotropic cytokine, initiates many aspects of the inflammatory response in the gastrointestinal mucosa (21, 22, 50). Whereas TNF-
acts as a growth factor for certain cell types (28), it has been implicated primarily in the induction of apoptosis through the activation of NF-
B and JNK depending on availability of downstream signaling proteins (5, 27, 37). MAPK family proteins are important transducers of cell signaling that coordinate the cellular response to various types of stimuli including TNF-
(34). The MAPK family consists of the ERKs, the JNKs, and the p38 kinases. JNK and p38 pathways are activated weakly in response to growth factors but are strongly activated in response to stress like UV radiation, reactive oxygen species, osmotic change, and TNF-
(31). Activation of JNK is correlated frequently with the apoptotic response (7, 33, 60). We showed that activation of the proapoptotic mediator JNK by TNF-
leads to apoptosis in IEC-6 cells. Polyamine depletion confers protection against the apoptotic assault by preventing the activation of JNK (6).
In contrast to the role of JNK, the ERK pathway is responsible primarily for cell growth and differentiation and is activated by phorbol esters, growth factors, and cytokines (8, 53). It has become evident in recent years that activation of MAPK can also lead to cell cycle arrest through the accumulation of p53 (18, 54). Studies from our laboratory have shown that -difluromethylornithine (DFMO) induced polyamine depletion in IEC-6 cells; increases ERK1/2 activity, p53 levels, and p21 levels; and leads to cell cycle arrest (49). TNF-
stimulates an increase in the tyrosine phosphorylation of ERK, which translocates to the nucleus and phosphorylates gene transactivators such as c-myc, c-Jun, and the serum response factor-1 involved in the regulation of cell proliferation and differentiation (8, 53). A growing number of reports point to the role of ERKs in mediating a strong antiapoptotic response (57, 62, 65). Activation of ERK protects cardiomyocytes from oxidative stress-induced cell death (1). ERK and NF-
B protect Jurkat cells from Fas-induced apoptosis (13, 23), and prevention of PC-12 cell death by N-acetylcysteine requires ERK activation (65). EGF, a potent activator of Ras-MAPK, counteracts IFN-
-mediated apoptosis in epidermal cancer cells (9). Hung et al. (24) recently reported that protection of renal epithelial cells against oxidative injury is mediated by ERK activation. The protective effect of activated ERK is mediated primarily through antiapoptotic members of the Bcl-2 family such as Bcl-2 and Bcl-xL (10, 41). ERK activation has been reported to inhibit caspase-8 cleavage and the translocation of Bax and, therefore, the release of cytochrome c (56).
We found that polyamine depletion of IEC-6 cells delays TNF- and camptothecin-induced apoptosis (48). Moreover, we have recently shown that TNF-
induces apoptosis in IEC-6 cells through a sustained activation of JNK. Polyamine depletion confers resistance to TNF-
-induced cell death by inhibiting JNK activation and cytochrome c release (6). Whether the inhibition of JNK activity in polyamine-depleted cells is concurrent with the activation of ERK is unknown. Moreover, the precise role of ERK in the regulation of apoptosis in intestinal epithelial cells has not been examined. A recently published report (55) demonstrated cross talk between JNK and ERK pathways. Given this background, we hypothesized that increased ERK activity in polyamine-depleted intestinal epithelial cells mediates the antiapoptotic response. Our results demonstrate that sustained activation of ERK in polyamine-depleted cells protects against apoptosis by inhibiting the JNK pathway.
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MATERIALS AND METHODS |
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Cell culture. IEC-6 cell stock was maintained in T-150 flasks in a humidified, 37°C incubator in an atmosphere of 90% air-10% CO2. The medium consisted of DMEM with 5% heat-inactivated FBS, 10 µg insulin, and 50 µg gentamicin sulfate per milliliter. The stock flask was passaged weekly, fed 3 times per week, and passages 15-22 were used. During experiment setup, the cells were trypsinized with 0.05% trypsin and 0.53 mM EDTA and counted by a Beckman Coulter Counter (model Z1). General protocols for experiments using IEC-6 cells have already been described (48). Briefly, IEC-6 cells were plated at 6.24 x 104 cells/cm2 in DMEM as described, fed every other day, and serum starved during the 24 h before harvesting. We (48) previously reported that maximal polyamine depletion occurs after 4 days of treatment with DFMO. Immediately after 6 h of DFMO treatment, putrescine was undetectable; spermidine was depleted after 24 h, and only 40% of spermine remained after 4 days.
Transfection. Transfection of IEC-6 cells has been described previously (47). Briefly, CA-hemagglutinin (HA)-pMCL, HA-8EK97, and pcDNA 3 (vector) DNA were prepared by using the Quiagen (Valencia, CA) (endotoxin-free) plasmid preparation kit. Fugene-6 reagent was mixed with DNA (3 µl:2 µg) in serum-free medium to a total volume of 100 µl and was incubated for 30 min at room temperature. IEC-6 cells at a relatively early passage were grown to 7080% confluence in 60-mm dishes. The antibiotic-resistant clones were trypsinized and grown to confluence in T-75 flasks in the presence of 200 µg/ml hygromycin B. Confluent cultures were trypsinized and plated at a very low density (1,000 cells per 100-mm culture plate), allowed to grow until they formed colonies, and cloned by using disposable cloning cylinders. Five clones for each were selected, seeded in T-75 flasks, and allowed to reach confluence in the presence of hygromycin B. Cell extracts were prepared from each clone and analyzed by Western blot for the presence of HA-tagged MEK protein. Further characterization of the clones was performed by immunohistochemical analysis of the HA-tagged protein in the MEK cells. IEC-6 cells were also transfected with the wild-type and DN-FLAG-tagged JNK constructs by using the same protocol. Clones were selected by 400 µg/ml of G418. Each clone was characterized by Western blot analysis with the FLAG and JNK antibodies.
Apoptosis studies. Cells were plated (day 0) at a density of 6.25 x 104 cells/cm2 in DMEM/dFBS with or without 5 mM DFMO or DFMO plus 10 µM putrescine, with triplicate samples for each group. Cells were fed on day 2. On day 3, the normal culture medium was removed and replaced with serum-free medium. On day 4, TNF- (20 ng/ml) with or without cycloheximide (CHX; 25 µg/ml) was added to the serum-free medium for 6 h, with the appropriate vehicle added to controls. Ten micromoles of U-0126 were added along with TNF/ CHX treatment with appropriate vehicle for inhibition of ERK1/2. For experiments involving camptothecin (CPT), the vehicle DMSO was used as a control, because CPT is reconstituted in DMSO. Ten micromoles of U-0126 were added along with CPT for inhibition of ERK1/2 phosphorylation.
Caspase activity and quantitative DNA fragmentation. After the induction of apoptosis by TNF-/CHX on day 4, floating cells were poured off and the monolayer was washed twice with cold Dulbecco's PBS. The attached cells were used for the determination of caspase activity and DNA fragmentation by using the cell death detection ELISA Plus kit as described earlier (6, 48, 67).
Mitochondrial permeability transition. Cells were grown for 4 days on 35-mm plates and treated with TNF- plus CHX for 1.5 h as described in Apoptosis studies. Cells were washed twice with 1x incubation buffer, and 1.0 ml of the diluted mitosensor reagent (final concentration, 5 µg/ml) was added on the plates and incubated at 37°C in 5% CO2 incubator for 20 min. Cells were gently rinsed with incubation buffer and examined under a fluorescent microscope by using a band-pass filter (detects fluorescein and rhodamine).
Western blot analysis. Cells were first washed with ice-cold PBS and lysed for 10 min in ice-cold extraction buffer. Cell extracts were analyzed by Western blot by using appropriate primary and secondary antibodies as described in our earlier publications (6, 48, 67). Blots were stripped and probed with the indicated antibodies to determine equal loading of the samples.
Kinase assays. ERK1/2 MAPK activity was determined by using a nonradioactive assay kit from Cell Signaling Technology following the manufacturer's instructions. Two-hundred micrograms of total protein was incubated with immobilized phospho-p44/42 MAP kinase (Thr202/Tyr204) monoclonal antibody with gentle rocking overnight at 4°C. Tubes were centrifuged for 30 s at 4°C and the pellet was washed twice with 500 µl lysis buffer, resuspended in 50 µl kinase buffer supplemented with 200 µM ATP and 2 µg Elk-1 fusion protein, and further incubated at 30°C for 30 min (39). The reaction was terminated with 25 µl SDS sample buffer, loaded on a 10% gel, transferred to a membrane, and probed with phospho-ELK1 antibody. The JNK assay was performed as described previously (32) by using a nonradioactive assay kit from Cell Signaling Technology. Briefly, cells were lysed as described above and 250 µg protein was transferred to separate tubes. Two micrograms (20 µl) of glutathione-S-transferase c-Jun fusion protein beads were added to the cell extracts and incubated overnight with gentle rocking at 4°C to pull down the stress-activated protein kinase (SAPK)/JNK. The pellet was centrifuged and washed four times. The pellet was suspended in 50 µl kinase buffer supplemented with 100 µM cold ATP. The reaction was terminated with 25 µl SDS sample buffer, boiled for 5 min, centrifuged for 2 min, and separated on a 10% SDS-PAGE. Phosphorylation of c-Jun was measured by using a phospho-c-Jun antibody.
Immunocytochemistry. Cells were seeded on coverslips coated with Matrigel and grown for 2 days. Semiconfluent cells were fixed in PBS/4% paraformaldehyde for 15 min at room temperature, followed by permeabilization in 0.1% Triton X-100 for 10 min and rinsed with PBS. Cells were blocked in sterile PBS with 2% BSA for 20 min. Subsequently, cells were rinsed with PBS/0.1% BSA for 20 min and incubated in the primary antibody (1:200) dilution of HA antibody at room temperature for 1 h. They were rinsed with PBS/0.1% BSA five times and incubated with FITC-conjugated secondary antibody for 1 h at room temperature. They were rinsed with PBS three times, mounted, and sealed. Images were captured by using a fluorescence microscope with a band-pass filter to detect fluorescein.
Statistics. All data are expressed as means ± SE from representative experiments. All experiments were repeated three times in triplicate. Analysis of variance and appropriate post hoc testing determined the significance of the differences between means. Values of P < 0.05 were regarded as significant.
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RESULTS |
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Effect of MEK inhibition. To establish the role of MAPK in TNF-/CHX-induced apoptosis in polyamine-depleted IEC-6 cells, we studied the role of the MAPK pathway by using a cell permeable MEK1/2 inhibitor, U-0126 (2, 16). To ensure that the MEK inhibitor actually inhibited TNF-
/CHX-induced ERK activity in these cells, we determined the levels of phosphorylated ERK protein by Western blot analysis. U-0126 almost totally inhibited the TNF-
/CHX-induced phospho-ERK1/2 protein (Fig. 2A). Polyamine-depleted cells had relatively higher levels of TNF-
/CHX-induced ERK1/2 activation compared with the control or cells grown with DFMO and putrescine. The MEK inhibitor completely inhibited ERK activation in control and DFMO plus putrescine groups, but the DFMO group showed detectable phospho-ERK. To ensure equal loading of the gel, the blot was stripped and probed with the ERK1/2 antibody. Figure 2 shows that there were no changes in the ERK protein level that could not be accounted for by phosphorylation.
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TNF-/CHX significantly increased DNA fragmentation compared with TNF-
alone. U-0126 had no effect by itself or in combination with TNF-
(Fig. 2B). Consistent with our previous observations (48), TNF-
/CHX treatment resulted in an approximate fivefold increase in DNA fragmentation compared with untreated cells, and addition of 10 µM U-0126 along with TNF-
/CHX did not change the level of DNA fragmentation (Fig. 3A). Polyamine depletion significantly attenuated the cytokine-induced DNA fragmentation (Fig. 3A), which is consistent with our earlier observation (48). The MEK inhibitor U-0126 sensitized the DFMO-treated cells and induced a significant DNA fragmentation (P < 0.005). However, U-0126 could not completely abolish the protective effect of polyamine depletion. The above results indicate that sustained activation of ERK (Fig. 1) in polyamine-depleted cells confers protection against apoptosis and that inhibition of MAPK eliminates some of their protection. The responses of cells treated with DFMO and putrescine were similar to those in the control group. U-0126 had no effect on TNF-
/CHX-induced JNK phosphorylation and activity in control cells, indicating that once JNK activation has reached a significant level, inhibition of ERKs does not reverse the onset of the apoptosis. On the contrary, polyamine depletion prevented JNK phosphorylation and activity (Fig. 3B). Addition of U-0126 along with TNF-
/CHX increased JNK phosphorylation and JNK activity in DFMO-treated cells (Fig. 3B). TNF-
/CHX induced processing of the 32-kDa procaspase-3 into its 20-kDa active form both in control cells and those treated with DFMO plus putrescine (Fig. 3). Consistent with our earlier observations, TNF-
/CHX could not activate caspase-3 in DFMO-treated cells. Addition of the MEK inhibitor abrogated the inhibitory effect of polyamine depletion on apoptosis and activated caspase-3 in these cells (Fig. 3B).
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To establish the correlation between the extent of ERK inhibition and augmentation of TNF-/CHX-induced apoptosis in polyamine-depleted IEC-6 cells, we examined the effect of increasing doses of U-0126. As shown in Fig. 4, there was an excellent correlation between U-0126-mediated ERK inhibition and dose-dependent caspase-3 activation. On the basis of the above observations, we believe that survival of polyamine-depleted cells is due to the prevention of JNK-mediated apoptotic pathways mediated by the MAPK pathway.
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Effect of CA-and DN-MEK1 expression on apoptosis. To further establish the role of MAPK in the suppression of apoptosis in polyamine-depleted cells, we overexpressed CA- and DN-MEK. Activation of MEK occurs through the phosphorylation of two key regulatory sites, Ser218 and Ser222. Substitution of glutamic or aspartic acid for Ser218 or Ser222 increases the basal activity of MEK four to eight times that of the wild-type enzyme (38). Both the CA and DN proteins were HA-tagged and have been characterized before (38). Clones of IEC-6 cells stably expressing CA- and DN-MEK1 were selected and characterized by immunohistochemical localization of the HA-tagged recombinant protein (Fig. 5A). IEC-6 cells transfected with the empty vector pcDNA31 showed no staining and were used as controls in all experiments. Figure 5B demonstrates the basal ERK phosphorylation and activity of the MEK transfected cells. Vector cells, CA and DN cells were grown to confluence in control conditions or in the presence of DFMO or DFMO plus putrescine. Cell lysates were analyzed for phospho-ERK by Western blots and ERK activity (kinase assay) by using ELK fusion protein as described in MATERIALS AND METHODS. Although basal ERK phosphorylation was undetected in vector cells, some degree of basal ERK activity was detected. On the other hand, CA-MEK1 cells had a high basal level of phospho-ERK1/2 and elevated ERK activity in control, DFMO-, and DFMO plus putrescine-treated cultures. DNMEK cells had no basal ERK-1 phosphorylation and low ERK activity compared with control vector cells or to the CA-MEK cells (Fig. 5B). ERK activity was stimulated only in response to polyamine depletion in DN-MEK cells, but there was almost no detectable ERK phosphorylation.
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Results depicted in Fig. 5, A and B establish the suitability of these clones to affirm the role of MEK in apoptosis. We then determined whether increased levels of ERK phosphorylation and activity correlated with a protective effect against TNF-/ CHX-induced apoptosis. Cells expressing DN-MEK1 had a significantly higher basal level of DNA fragmentation, which was inhibited by polyamine depletion. Exogenous addition of putrescine prevented the effect of DFMO. TNF-
/CHX treatment further increased DNA fragmentation in the DN-MEK cells. Polyamine depletion partially protected the DN-MEK1 cells from TNF-
/CHX-induced apoptosis. Cells transfected with CA-MEK and empty vector showed no significant differences in basal apoptosis. TNF-
/CHX treatment induced apoptosis in vector-transfected cells grown either in control conditions or in the presence of DFMO and putrescine. Similar to that observed in normal IEC-6 cells, polyamine depletion protected empty vector cells from apoptosis. TNF-
/CHX-induced DNA fragmentation was significantly reduced in CAMEK cells grown both in control conditions and in the presence of DFMO plus putrescine (Fig. 6A). Polyamine depletion of the CA-MEK cells inhibited DNA fragmentation compared with vector cells. TNF-
/CHX induced phosphorylation of ERK in vector and CA-MEK cells, whereas only DFMO-treated DN-MEK cells showed ERK phosphorylation (Fig. 6B), which is consistent with decreased apoptosis compared with cells grown both in control conditions and in the presence of DFMO plus putrescine (Fig. 6A).
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Consistent with the DNA fragmentation data presented in Fig. 6A, there was little difference in caspase-3 activity between vector and CA-MEK cells, and TNF-/CHX had no significant effect on caspase activation in the CA-MEK cells (Fig. 7A). Compared with vector cells, caspase-3 activity was high under basal conditions in DN-MEK cells and could not be increased significantly by TNF-
/CHX (Fig. 7B). Polyamine depletion lowered caspase-3 activity significantly in vector cells but had no effect in the DN-MEK group. These results clearly indicate that activation of the MAPK pathway rescues cells from TNF-
/CHX-induced apoptosis, and inhibition of this pathway by DN-MEK can render the cells more susceptible to apoptosis. The failure of DFMO to rescue DN-MEK cells from apoptosis is evidence that the protection normally afforded by polyamine depletion is mediated through ERK1/2.
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Correlation of JNK activation with apoptosis in CA-MEK and DN-MEK cells. Because inhibition of ERK by U-0126 activated JNK and increased apoptosis in polyamine-depleted cells, we examined JNK activity in IEC-6 cells transfected with CA- or DN-MEK. TNF-/CHX induced JNK phosphorylation and JNK activity in vector, CA-, and DN-MEK1 cells (Fig. 8). Consistent with our previous observations (Fig. 3), polyamine depletion inhibited TNF-
/CHX-induced phosphorylation of both p54 and p46 JNK and JNK activity in vector cells compared with cells grown under control conditions or with DFMO plus putrescine.
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JNK phosphorylation and JNK activity induced by TNF-/ CHX was significantly inhibited in CA-MEK cells compared with the vector cells. Polyamine depletion completely prevented TNF-
/CHX-induced JNK activation in the CA-MEK1 cells. Conversely, DN-MEK1 cells grown either in control conditions or in the presence of DFMO plus putrescine had high basal levels of phosphorylation of both JNK-p54 and -p46 isoforms. Interestingly, basal levels of JNK phosphorylation and activity were observed in polyamine-depleted DN-MEK cells, indicating some loss of protection by DFMO. Treatment of these cells with TNF-
/CHX did not significantly increase the level of phospho-JNK or JNK activity. The finding that polyamine depletion did not inhibit TNF-
/CHX-induced JNK activation in DN-MEK1 cells indicates that ERK1/2 activation plays a pivotal role in the antiapoptotic effects of polyamine depletion.
We have previously reported (6) that TNF-/CHX-induced apoptosis is mediated through JNK activation, which eventually leads to the release of cytochrome c and activation of caspase-9 and -3. Polyamine depletion inhibited JNK activation and the release of cytochrome c (6). We used Mitosensor dye to detect cytochrome c release. Mitosensor aggregates in the mitochondria of healthy cells and fluoresces red. In apoptotic cells, mitochondrial membrane potentials are altered and the dye cannot accumulate in mitochondria. It remains as a monomer in the cytoplasm and fluoresces green. TNF-
/CHX induced cytochrome c release in all three groups of vector cells, but there was significantly less release from cells depleted of polyamines than from controls and those grown with DFMO and putrescine (Fig. 9A). The DN-MEK cells had an increased level of basal release, indicating a high level of apoptosis (Fig. 9B). TNF-
/CHX did not significantly increase the basal level of cytochrome c release, and DFMO had no discernible effect on cytochrome c release in these cells (Fig. 9B).
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DN-JNK1 decreases apoptosis. Data generated so far clearly indicate that sustained ERK1/2 activation in DFMO-treated cells inhibits JNK activity and decreases apoptosis. To substantiate the role of JNK activation as a central mediator of apoptosis, we transfected IEC-6 cells with FLAG-tagged wild-type JNK1 (WT-JNK1) or with the DN-JNK1 constructs. Figure 10A shows expression of FLAG tag. Empty vector (without FLAG tag)-transfected IEC-6 cells were used as controls. TNF-/CHX induced a significant level of DNA fragmentation in all three cell types compared with untreated cells. However, the degree of apoptosis was significantly less in the DN-JNK1 cells (Fig. 10B). TNF-
/CHX did not induce JNK activation in polyamine-depleted cells unless MEK1 was inhibited by U-0126. Because JNK is downstream from MEK1 and because it is already inhibited in polyamine-depleted cells, we speculate that inhibition of MEK1 in polyamine-depleted DN-JNK1 cells should not sensitize them to apoptosis. As predicted, inhibition of MEK1 by U-0126 did not increase DNA fragmentation in DFMO-treated DN-JNK1 cells (data not shown). These results support our conclusion that activation of JNK is an integral part of the apoptotic response in IEC-6 cells.
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Inhibition of ERK phosphorylation reverses protection from camptothecin-induced apoptosis in polyamine-depleted IEC-6 cells. Earlier, we (67) reported that the DNA topoisomerase inhibitor CPT induced apoptosis in IEC-6 cells, which was prevented by polyamine depletion. Because our current data showed that inhibition of MEK1 reversed the protective effect of polyamine depletion during TNF-/CHX-induced apoptosis, we tested whether sustained activation of ERK plays a central role in the protection of IEC-6 cells from a nonreceptor-mediated apoptotic stimulus. Exposure to CPT for 6 h induced ERK activation in all three groups of cells (Fig. 11A). Polyamine-depleted cells had the highest level of ERK phosphorylation in response to CPT. The MEK inhibitor significantly prevented CPT-induced ERK phosphorylation in all three groups. CPT increased JNK phosphorylation in all three groups, but the increase was considerably less in the DFMO group. Inhibition of MEK had little effect on JNK phosphorylation in control cells and cells grown with DFMO and putrescine but significantly increased phosphorylation in the polyamine-depleted cells. In general, the apoptotic responses to CPT were identical to those to TNF-
/CHX under the same conditions. CPT increased DNA fragmentation in all three groups, but there was considerable protection in the polyamine-depleted cells and the level of DNA fragmentation was considerably less. Inhibition of MEK significantly increased DNA fragmentation in the DFMO group but had no significant effect on the amount of apoptosis in the control and DFMO plus putrescine group (Fig. 11B). Nearly identical results with TNF-
/CHX were shown in Fig. 3B. These results indicate that ERK activation is pivotal in conferring resistance to various apoptotic stimuli in polyamine-depleted cells. Interference with the activity of this protein in DFMO-treated cells renders them susceptible to apoptosis.
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DISCUSSION |
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An earlier report from our laboratory (48) showed that apoptosis induced by both the DNA-damaging agent camptothecin and by TNF- plus CHX was delayed in polyamine-depleted cells, suggesting an important role for polyamines in this process. However, pathways influenced by polyamines in the apoptosis of intestinal epithelial cells are poorly understood. Recently, we established that JNK activation in response to TNF-
/CHX induced apoptosis via cytochrome c release and that polyamine depletion prevented JNK activation and apoptosis (6).
Growing evidence in the literature suggests a role for ERK1/2 activation in the protective response against oxidative stress and apoptosis (1, 9, 23, 24, 56, 57). TNF- initiates a kinase cascade in which MEKK activates MEK, thereby causing tyrosine phosphorylation and activation of p42/p44 MAPK/ ERK1/2 (51). This study examined the role of MAPK family proteins in the regulation of apoptotic signaling in normal and polyamine-depleted IEC-6 cells. P38, an MAPK family member widely implicated in apoptosis, was not correlated with the apoptotic response in IEC-6 cells (data not shown). Interestingly, findings that TNF-
and CHX alone and in combination induced ERK activation indicated a role for ERK in the process of apoptosis. Increasing evidence has also demonstrated both the pro- and antiapoptotic nature of ERK signaling, depending on cell type and stimulus (9, 23, 25, 42). Our results demonstrate that ERK activation plays a significant role in the apoptotic response of intestinal epithelial cells. More importantly, a time-dependent, sustained, and potent activation of ERK1/2 indicated an important role for ERK signaling in the antiapoptotic response to polyamine depletion.
We further established the antiapoptotic role of MAPK signaling by using the membrane-permeable MEK inhibitor U-0126, which significantly inhibited TNF-/CHX-mediated ERK activation (Fig. 2A). Inhibition of MAPK sensitized polyamine-depleted cells to apoptosis, which otherwise were resistant to the apoptotic stimulus. MEK inhibitors U-0126 and PD-98059 have been previously reported to sensitize cells to apoptosis (13, 30). Figure 2B shows that U-0126 or U-0126 plus TNF-
did not increase DNA fragmentation, indicating that the dose of U-0126 used in this study had no cytotoxic effects and that the levels of ERK activation in response to TNF-
/CHX play an important role in the regulation of apoptosis. Addition of the MEK inhibitor at the time of TNF-
/ CHX administration significantly reversed the protective effect of DFMO (Fig. 3). However, addition of the MEK inhibitor with TNF-
/CHX had no significant effect on apoptosis in control cells or those grown with DFMO and putrescine (Fig. 3A). These findings are explained by the data shown in Figs. 1 and 3B. TNF-
/CHX caused an immediate activation of JNK in control cells and in cells to which polyamines had been added exogenously. There was no significant activation of JNK in polyamine-depleted cells (Fig. 3B). Figure 1 shows that TNF-
/CHX trigger a strong activation of ERK in all three groups of cells. In the case of control cells, ERK activation begins to decrease by 6 h, whereas in polyamine-depleted cells, ERK activation continues to increase with time. In our recent previous study (6), JNK activity in response to TNF-
/CHX in control cells increased at 3 h and continued to increase with time, unlike ERK activity under the same conditions. These observations clearly indicate that the amount of initial ERK1/2 activation is not sufficient to prevent JNK activation. It also has been reported that caspase activation-mediated feedback can enhance apoptosis by increasing JNK activation (14, 36). Thus it is clear from our study that the degree of activation of ERK modulates the level of JNK activity, which is a key mediator of apoptosis in our IEC-6 cells. Thus polyamine-depleted cells increase ERK activation with time, maintaining a basal level of JNK phosphorylation, and inhibition of ERK activation by U-0126 in these cells increases DNA fragmentation with a concomitant increase in JNK phosphorylation and, thereby, JNK activity (Fig. 3).
Our previous study (6) also showed that SP-60125, a specific inhibitor of JNK phosphorylation, prevented apoptosis in control cells by inhibiting cytochrome c release and the subsequent activation of caspase-9 and -3. Mitochondrial pathways play a critical role in the regulation of apoptosis. Cytochrome c is located in the intermembrane space of the mitochondria and is released into the cytosol in response to apoptotic agents (29, 35). The cellular apoptotic machinery responsible for cytochrome c release is regulated by the family of Bcl-2 proteins, which mainly involves the death agonists Bax, Bak, Bad, and Bcl-xS and antagonists Bcl-2, Bcl-w, Mcl-1, and Bcl-xL (4, 15). Bcl-2 regulates apoptosis by interacting with proapoptotic proteins Bax and Bad and inhibiting cytochrome c release from mitochondria (52, 66).
Increasing evidence suggests the participation of activated ERK in the mitochondrial pathway through the regulation of Bcl-2 mRNA and protein levels and subsequent cytochrome c release (10, 30, 41, 56). Allan et al. (3) reported that phosphorylation of ERK inhibited caspase-9. Inhibition of ERK1/2 activity by U-0126 and the resulting increased JNK activation and caspase-3 processing in DFMO-treated cells suggest that the protection afforded by polyamine depletion is due to the balance between ERK activity and JNK phosphorylation. In addition, Fig. 4 shows a close relationship between caspase-3 activation and ERK1/2 inhibition in response to increasing concentrations of U-0126 in polyamine-depleted cells. These results indicate the possibility that ERK1/2 may regulate an upstream activator of JNK and subsequent downstream signaling events leading to apoptosis in IEC-6 cells. MKP7, a JNK-specific phosphatase, regulates JNK phosphorylation and is induced after activation of ERK, and inhibition of MKP7 has been shown to sensitize cells to apoptosis (40). We speculate that ERK or an upstream regulator may be responsible for the regulation of basal JNK activity and survival of cells after apoptotic signals. Shen et al. (55) recently reported cross talk between JNK/SAPK and ERK/MAPK pathways, and opposing effects of JNK and MAPK/ERK in apoptosis have been reported previously (62).
CA-MEK1-expressing cells with higher ERK1/2 activity before the administration of an apoptotic signal were protected from TNF-/CHX-induced apoptosis, and DN-MEK1 cells with low ERK1/2 activity showed higher basal and TNF-
/ CHX-induced DNA fragmentation and caspase-3 activity (Figs. 6 and 7). Furthermore, DN-MEK-1 cells had significantly high levels of basal and TNF-
/CHX-induced JNK phosphorylation compared with vector and CA-MEK1 cells. Subsequent increased cytochrome c release in DN-MEK cells clearly indicates a role for MEK-1-mediated ERK1/2 activation and JNK phosphorylation in IEC-6 cells (Figs. 8 and 9). Suppression of apoptosis in the CA-MEK cells may be due to increased synthesis of antiapoptotic proteins stimulated by ERK before the addition of TNF-
and CHX. DNA fragmentation was significantly decreased in DN-JNK1 compared with WT-JNK1 cells or vector cells (Fig. 10). Thus our results with MEK-1 and JNK-transfected cells further confirmed our hypothesis regarding the role of ERK1/2 and JNK activation in the regulation of apoptosis and that the effect of polyamine depletion is exerted upstream of JNK activation by regulating ERK activity. Current findings using the chemical inhibitor U-0126 and a DN mutant of MEK demonstrated an essential role for MEK1 in apoptosis suppression (20). Kurland et al. (30) recently showed that ERK1 and ERK2 were constitutively phosphorylated in a murine B cell lymphoma cell line that was refractory to apoptosis, whereas a cell line that was sensitive to apoptosis had no detectable levels of phosphorylated ERK1 or ERK2. In the same study (30), the inhibition of MEK sensitized cells, previously refractory to apoptosis, to radiation-induced apoptosis by regulating Bcl-2 expression.
Finally, to determine whether the mechanism of protection by polyamine depletion, elucidated here for TNF-, applied to other inducers of apoptosis we tested CPT, a non-receptor-mediated apoptotic inducer. Earlier, we (6) reported that CPT induced apoptosis in IEC-6 cells via cytochrome c, Bcl-2, Bax, and Bad and that polyamine depletion delayed it by inhibiting the mitochondrial pathway and in turn caspase-8, -9, and -3. In the present study, we showed that CPT-induced apoptosis also involved JNK activation. U-0126, the specific inhibitor of MEK1/2, significantly increased DNA fragmentation and JNK activation in polyamine-depleted cells without significant effects in control cells and those grown with DFMO and putrescine. Thus it appears that JNK activation plays a central role in the process of apoptosis via cytochrome c release and that polyamines regulate JNK activation by regulating MEKERK1/2. JNK was shown recently to be a stress-activated Bcl-2 kinase, which inactivates Bcl-2 by phosphorylating it (11), and DN-JNK prevents Bcl-2 phosphorylation (64). Kurland et al. (30) found that the MEK/ERK pathway acts upstream of both the NF-
B and Bcl-2 survival pathways, implicating the MAPK pathway as a key upstream mediator of apoptosis.
We conclude that the MAPK pathway regulates apoptosis by modulating JNK activation and, in turn, cytochrome c release and the caspase cascade. In the absence of polyamines, sustained activation of ERK1/2 prevents JNK activation. The MAPK-mediated resistance to apoptosis in polyamine-depleted cells may be due to 1) upregulation of a JNK-specific phosphatase like MKP-7, 2) an ERK-mediated increase in the level of Bcl-2 and prevention of Bcl-2 phosphorylation by JNK, or 3) the ERK-mediated expression of antiapoptotic proteins.
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ACKNOWLEDGMENTS |
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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-16505 and by the Thomas A. Gerwin Endowment.
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FOOTNOTES |
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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.
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REFERENCES |
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