Am J Physiol Gastrointest Liver Physiol 293: G599-G606, 2007.
First published July 5, 2007; doi:10.1152/ajpgi.00182.2007
0193-1857/07 $8.00
MUCOSAL BIOLOGY
Epidermal growth factor receptor signaling modulates apoptosis via p38
MAPK-dependent activation of Bax in intestinal epithelial cells
George Sheng,
Jun Guo, and
Brad W. Warner
Division of Pediatric General and Thoracic Surgery, Cincinnati Children's Hospital Medical Center, Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, Ohio
Submitted 25 April 2007
; accepted in final form 1 July 2007
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ABSTRACT
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Previous studies have demonstrated that the proapoptotic protein Bax plays an important role in the elevated enterocyte apoptosis that occurs during the intestinal adaptation response to massive small bowel resection (SBR). Additionally, epidermal growth factor receptor (EGFR) activation prevents SBR-induced enterocyte apoptosis. The present study aims to delineate the relationship between EGFR activity and intestinal epithelial cell apoptosis. Treatment of model intestinal epithelial cells (RIEC-18) with both a selective EGFR inhibitor (ZD1839) and EGFR small interfering RNA knockdown resulted in a dramatic increase in apoptosis, accompanied by rapid phosphorylation of p38
. Concurrently, Bax underwent conformational changes consistent with activation and translocated to mitochondria. In contrast, EGF stimulation enhanced cell survival by attenuating p38 phosphorylation, Bax conformational change, mitochondrial trafficking, and apoptosis. These results demonstrate that that diminished EGFR activity initiates the intrinsic pathway of apoptosis through p38-dependent Bax activation in intestinal epithelial cells. These finding provide mechanistic insight into the role that EGFR signaling plays in the regulation of enterocyte apoptosis following massive intestinal loss.
intestinal adaptation; short bowel syndrome
FOLLOWING THE RESECTION OF significant intestinal length as required to treat multiple conditions such as midgut volvulus, trauma, or necrotizing enterocolitis, the remaining bowel undergoes a crucial compensatory response termed adaptation. During this process, there is a stimulus for enterocyte proliferation that contributes to the generation of taller villi, deeper crypts, and a greater content of DNA, RNA, and protein per unit length (5, 17). Through these morphological alterations, the remaining intestine attempts to compensate for the loss of mucosal surface area by increasing its capacity for absorption and digestion per unit length.
In addition to increased rates of enterocyte proliferation, our laboratory was the first to demonstrate enhanced enterocyte apoptosis during resection-induced adaptation (13). This observation has been confirmed by other investigators (12, 22–24). Given the highly dynamic nature of the intestinal mucosa, elevated rates of apoptosis probably serve to counter the increased rates of enterocyte proliferation, thus preserving a critical homeostatic balance. Although much attention has focused on understanding the regulation of enterocyte proliferation, few studies have addressed the mechanism for increased apoptosis during this process.
Members of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases serve as critical mediators of the cellular communication network regulating complex biological processes such as growth, differentiation, motility, and death (11, 20). Over the past decade, our laboratory has illuminated a central role for EGFR signaling in the genesis of resection-induced adaptation (26). In addition to affecting rates of enterocyte proliferation, EGFR activity was also found to regulate enterocyte apoptosis after massive small bowel resection (SBR) (14, 18). In wave-2 mice with hypofunctional EGFR activity, apoptosis is markedly enhanced both at baseline and following a massive SBR (14, 15). Conversely, stimulation of the EGFR either by exogenous administration of EGF or in EGF transgenic mice is associated with diminished apoptotic response to SBR (14).
An important class of molecules that regulate apoptosis within complex mammalian cells is the Bcl-2 family of intracellular proteins. By performing intestinal resections in Bax-null mice, we established that Bax was the proapoptotic Bcl-2 family member required for resection-induced enterocyte apoptosis (1, 21). Despite strong evidence for both EGFR signaling and Bax as major regulators of enterocyte apoptosis, a mechanistic link between these two factors is poorly characterized. This is the very basis for the present study.
The p38 mitogen-activated protein kinase (MAPK) is a member of the MAPK pathway and is activated in response to various of physical and chemical stresses, such as oxidative stress, UV irradiation, hypoxia, ischemia, and various cytokines, including interleukin-1 (IL-1) and tumor necrosis factor- (3). Additionally, it has been shown that p38 phosphorylation plays an important role in enterocyte migration and EGFR degradation following stimulation (6). In preliminary studies from our laboratory, we have revealed that p38 MAPK phosphorylation and activity were significantly enhanced within intestinal crypts following massive SBR. This finding coincided with elevated enterocyte apoptosis in the crypts and suggested a potential role for p38 in resection induced enterocyte apoptosis.
Until recently, the potential interaction between p38 and Bcl-2 family has been poorly understood. A recent study suggested that p38 was required to induce a conformational change in Bax, thereby allowing for its translocation to mitochondria and cytochrome c release following UV damage (25). Our present study attempted to explore the relationship between EGFR modulation of Bax-dependent enterocyte apoptosis via p38, thus providing a novel mechanism for EGFR-regulated enterocyte apoptosis after SBR. Since our in vivo experimental model focuses on physiological processes in normal enterocytes, it is particularly relevant to study the relationship between EGFR signaling and its effect on enterocyte apoptosis by using nontransformed cells. We chose IEC-18 because it is a nontransformed cell line derived from rat terminal ileum and corresponds with the same anatomical location where we observe maximal responses to resection-induced intestinal adaptation.
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MATERIALS AND METHODS
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Cell culture and materials.
IEC-18 cells from ATCC were cultured in DMEM supplemented with 10% fetal bovine serum, 100 unit/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine. Cells used in experiments were between passage 15 and 20. The EGFR inhibitor ZD1839 was obtained from AstraZeneca (Wilmington, DE). Another EGFR inhibitor, AG1478, and p38 inhibitors SB203580 and SB239063 were purchased from Biosource (Camarillo, CA). Monoclonal anti-Bax 6A7 antibody was purchased from BD Pharmingen (San Diego, CA). Polyclonal anti-EGFR antibody directed against mouse EGFR was generated with a synthetic peptide corresponding to residues 1158–1175 of mouse EGFR as the immunogen. This sequence is unique to EGFR and is identical in rat and mouse, conserved in human and mouse (95% identity). The anti-EGFR serum from rabbit was further affinity purified against the original peptide and specificity tested by Western blot. Polyclonal Bax and phosphorylation-specific p38 antibodies were from Cell Signaling (Beverly, MA).
Western blot and immunoprecipitation.
IEC-18 cells were lysed in 1x SDS sample buffer (50 mM Tris·HCl pH 6.8, 2% SDS, 10% glycerol, and 5% mercaptoethanol). For immunoprecipitation, the cells were washed with ice-cold PBS and then lysed for 30 min in lysis buffer containing 10 mM HEPES, 150 mM NaCl, and 1% CHAPS plus protease inhibitor tablet (Roche, Indianapolis, IN) and phosphatase inhibitor cocktail I/II (Sigma). The lysates were then centrifuged at 20,000 g for 30 min at 4°C. The supernatant were then incubated with protein A plus G beads (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h (preclearing) at 4°C. Following this, the beads were spun down and discarded, and the supernatants were further incubated with protein A plus G beads and appropriate antibody overnight at 4°C. After being washed in lysis buffer, the absorbed complexes were removed from the beads by heating for 5 min at 100°C in 1x SDS sample buffer and separated on 10% polyacrylamide gels. Proteins resolved on gels were transferred to nitrocellulose membranes and detected with appropriate antibodies by Western blot.
Apoptosis assay.
Apoptosis was measured in cells by using an apoptosis ELISA kit (Roche) according to the protocol of the manufacturer. Briefly, IEC-18 cells were induced to undergo apoptosis according to the experiment. Cells were lysed and fragmented DNA was separated from intact DNA by centrifugation. The amount of fragmented DNA was recognized by a specific antibody against DNA binding proteins and results analyzed by ELISA.
Immunocytochemistry.
IEC-18 cells were grown on coverslips in six-well culture plates. Following induction of apoptosis, cells were incubated with 250 nM of MitoTsracker Red (Cambrex, Walkersville, MD) for 15 min and then fixed with 4% formalin in PBS and permeablized with 1% CHAPS. After being blocked with 3% BSA cells were incubated with 1:300 of Bax (6A7) antibody for 1 h in 37°C incubator. After three washes, cells were incubated for 45 min in secondary antibody. Cell nuclei were stained with DAPI for 10 min, slides were mounted, and images were recorded under x100 and x400 magnification.
siRNA transfection.
Roughly 1 x 106 IEC-18 cells were transfected with EGFR, p-38 or Bax small interfering RNA (siRNA; SMARTpool reagent; Lafayette, CO). Control was provided by transfection with a nontargeting RNA (siCONTROL nontargeting siRNA; Dharmacon; Lafayette, CO) at 25 nM concentration with X-tremeGENE (Roche), according to the manufacturer's protocol. Cells were collected 72 h after transfection and used for experiments. For EGFR silencing, EGFR siRNAs were transfected into IEC-18 cells at three different concentrations (10, 15, and 25 nM). To confirm silencing, cells were collected and Western blots were performed to measure target protein expression.
Mitochondrial isolation.
For each experiment, 5 x 107 cells were collected. Mitochondrial and cytosolic fractions were separated by using a Mitochondrial fractionation kit (Active Motif, Carlsbad, CA). After fractionation, protein concentration in each fraction was measured. Western blot analysis of each fraction was performed.
Statistics.
For comparisons of mean values between two groups, Student's t-test was used. For comparisons of mean values between more than two groups, a one-way analysis of variance was employed. Statistical significance was established at a P value of <0.05.
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RESULTS
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EGFR activity modulates apoptosis in intestinal epithelial cells.
To study the apoptotic response of intestinal epithelial cells with regard to EGFR activity, IEC-18 cells were treated with either EGF or with EGFR inhibitors (ZD1839 or AG1478) at different concentrations. Inhibition of the EGFR was additionally accomplished by siRNA-directed suppression of EGFR expression. Compared with control, EGF stimulation resulted in dose-dependent increases in EGFR phosphorylation and attenuated levels of apoptosis (Fig. 1A). To ensure that EGFR inhibitor-induced apoptosis was not specific to ZD1839, another selective EGFR inhibitor (AG1478) was shown to induce apoptosis in IEC-18 cells in dose-dependent fashion (Fig. 1B). Furthermore, knockdown of EGFR protein expression using siRNA resulted in dose-dependent increase in RIEC-18 apoptosis (Fig. 1C).
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Fig. 1. A: IEC-18 cells were grown to 80% confluence in 1% FBS in DMEM. To inhibit EGF receptor (EGFR) activity, 2 different concentrations of ZD1839 were used. To enhance EGFR activity, 5 different concentrations of EGF were used to stimulate cells. Cells were harvested 4 h after treatment, and apoptosis was measured by DNA fragmentation ELISA. Results were plotted as fold changes over vehicle (DMSO)-treated cells. Con, control. B: IEC-18 cells were treated with AG1478, a selective EGFR inhibitor, at varied concentrations. Apoptosis was measured 4 h later by DNA fragmentation ELISA and results displayed as fold changes over vehicle (DMSO)-treated cells. C: IEC-18 cells were grown to 50% confluence and transfected with EGFR small interfering RNA (siRNA) at 3 different concentrations for 3 days. Control (scrambled) siRNA were also transfected into a separate group of cells for experimental control. Apoptosis was measured 4 h later by DNA fragmentation ELISA and results were displayed as fold changes over vehicle (DMSO)-treated cells. *P < 0.05 vs. control.
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EGFR inhibition-induced apoptosis is associated with activation of p38 MAPK and Bax.
Treatment of the cells with the EGFR inhibitor ZD1839 (10 µM) resulted in a significant reduction in baseline EGFR activity as gauged by its phosphorylation status (Fig. 2A). Concurrently, phosphorylation of p38 increased during EGFR inhibition whereas total p38 protein expression remained unchanged. Inhibition of the EGFR was also accompanied by accumulation of the conformationally active form of Bax (6A7) but without change in the expression of native Bax. Cleaved caspase-3 was also elevated and corresponded with the induction of apoptosis. Knockdown of EGFR expression using siRNA similarly resulted in a concentration-dependent suppression of EGFR expression, increased p38 phosphorylation, and activation of Bax (Fig. 2B). Alternatively, stimulation with EGF resulted in dose-dependent phosphorylation of the EGFR, decreased phosphorylation of p38, reduction in Bax activity, as well as diminished cleaved caspase-3 accumulation (Fig. 2C).
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Fig. 2. A: IEC-18 cells were harvested at different time points following treatment with ZD1839 (10 µM). Expressions of tyrosine-phosphorylated EGFR, p38, phosphorylated p38, caspase-3, cleaved caspase-3, and Bax were measured by Western blot. Bax (6A7) antibody was used to immunoprecipitate conformationally active Bax from the remaining lysates followed by Western blot quantification. B: IEC-18 cells were grown to 50% confluence and transfected with EGFR siRNA at 3 different concentrations for 3 days. Control (scrambled) siRNA (sicon) were also transfected into a separate group of cells for experimental control. Cells were harvested after 3 days of transfection and proteins harvested for Western blot analysis of EGFR, p38, phospho-p38, and Bax. Immunoprecipitations were performed for conformationally active Bax. Apoptosis of IEC-18 cells following EGFR silencing were performed by DNA fragmentation ELISA. C: IEC-18 cells were harvested 4 h following stimulation with different concentrations of EGF. MAPK, caspase-3, cleaved caspase-3, and Bax were measured by Western blot. Bax (6A7) antibody was used to immunoprecipitate conformational active Bax from the remaining lysates followed by Western blot quantification.
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EGFR inhibition induces mitochondrial trafficking of activated Bax.
Bax activation results in initiation of the intrinsic apoptotic pathway by increasing mitochondrial permeability and release of cytochrome c. Following EGFR inhibition, activated Bax became concentrated within the mitochondrial fraction and was reduced in the cytosolic fraction (Fig. 3A). Additionally, cytosolic cytochrome c was increased, consistent with increased release from the mitochondria. The mitochondrial trafficking of activated Bax was confirmed by immunocytochemistry (Fig. 3B). Together, these findings confirm that EGFR inhibition leads to the activation of the intrinsic apoptotic pathway via trafficking of activated Bax to the mitochondria.
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Fig. 3. A: IEC-18 were grown to 80% confluence, and after overnight growth in 1% FBS cells were treated with ZD1839 for 2, 4, and 6 h. Mitochondrial and cytosolic fractions were separated by use of a mitochondrial isolation kit. Western blot for Bax and cytochrome c were performed on different fractions. B: IEC-18 cells were treated with ZD1839 for 4 h. Immunocytochemistry was performed to detect conformational active Bax with Bax (6A7) antibody. Mitochondrial stain was done by incubation with MitoTracker RED 45 min prior to fixation. Nuclei were stained by DAPI. Intracellular locations of Bax were demonstrated by image overlay.
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Bax expression is required for induction of apoptosis by EGFR inhibition.
To verify that Bax is necessary for the induction of apoptosis associated with EGFR inhibition, Bax expression was suppressed in IEC-18 cells by use of siRNA. Significant knockdown of Bax expression was achieved by siRNA when compared with control siRNA-transfected cells (Fig. 4A). The cells with attenuated Bax expression were resistant to apoptosis induced by pharmacological inhibition of the EGFR when compared with cells with normal levels of Bax (Fig. 4B). Nonetheless, p38 phosphorylation in response to EGFR inhibition was not affected in the context of reduced Bax expression (Fig. 4A). These findings support the notion that Bax is required for the apoptotic response to EGFR inhibition. Furthermore, these data suggest that perturbed EGFR activity induces p38 activation, which in the context of attenuated Bax expression is insufficient to trigger apoptosis.
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Fig. 4. A: Bax expression was suppressed by siRNA transfection for 3 days. After confirmation of gene knockdown by Western blot, cells were treated with ZD1839 (10 µM). Effect of Bax expression knockdown on p38 phosphorylation and Bax activation were evaluated with Western blot and immunoprecipitation, respectively. Initiation of apoptosis was measured by cleaved caspase-3 expression. B: DNA fragmentation ELISA was performed at the same time to evaluate cell death. *P < 0.05 vs. cells not treated with ZD (–ZD).
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EGFR inhibition induces specific p38 subunit phosphorylation.
The p38 MAPK has four distinct isoforms (, 
, 
, and 
). To determine the most important isoform, phosphorylated p38 was immunoprecipitated after EGFR inhibition and specific isoforms were analyzed by Western blotting. As shown in Fig. 5, all four isoforms were detectable in IEC-18 cells; however, only the -subunit was present within the pulled-down phosphorylated fraction of p38. These findings strongly suggest that the p38 isoform is the most relevant downstream target of EGFR inhibition.
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Fig. 5. Following treatment of IEC-18 cells, phosphorylated p38 was immunoprecipitated (IP). Different isoforms of p38 were quantified by Western blot. Western blot analysis of total p38 was used to confirm equal loading.
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Inhibition of p38 MAPK results in reduced Bax activity and apoptosis.
To explore a potential link between p38 phosphorylation and Bax activation, IEC-18 cells were pretreated with a selective inhibitor of p38 (SB203580) prior to EGFR inhibition. The results revealed that when p38 phosphorylation was pharmacologically inhibited, EGFR inhibition failed to initiate a significant change in Bax activation (Fig. 6A). Furthermore, the apoptotic response to EGFR inhibition was greatly reduced.
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Fig. 6. A: IEC-18 cells were pretreated with SB203580 (SB; 10 µM) for 1 h followed by ZD1839 treatment (ZD; 10 µM) for 4 h. Apoptosis was measured by DNA fragmentation ELISA. Bax activation was measured by immunoprecipitation of conformational active Bax (6A7) followed by Western blot analysis of Bax. B: IEC-18 cells were pretreated with SB203580 (10 µM) for 1 h followed by ZD1839 treatment (10 µM) for 4 h. Immunocytochemistry was performed to detect conformational active Bax with Bax (6A7) antibody. Mitochondrial stain was done by incubation with MitoTracker Red 45 min prior to fixation. Intracellular locations of Bax were demonstrated by image overlay. C: P38 expression was suppressed by siRNA transfection for 3 days. After confirmation of gene knockdown by Western blot, cells were treated with ZD1839 (10 µM). Effect of p38 expression and phosphorylation knockdown on Bax activation was evaluated with immunoprecipitation of active Bax. Initiation of apoptosis was measured by cleaved caspase-3 expression. D: DNA fragmentation ELISA was performed at the same time to evaluate cell death. *P < 0.05 vs. cells not treated with inhibitor (–ZD).
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Pharmacological inhibition of p38 prevents Bax translocation to mitochondria.
As shown earlier, conformationally active Bax translocated to mitochondria following EGFR inhibition. Using immunocytochemistry, we now show that when p38 activity was inhibited pharmacologically, the perturbed EGFR signal to direct mitochondrial translocation of Bax was greatly reduced (Fig. 6B).
P38 is required for Bax activation and apoptotic response following EGFR inhibition by ZD1839.
Having established that EGFR inhibitor-induced apoptosis in IEC-18 cells involved phosphorylation of p38 as well as activation and translocation of Bax, we next silenced p38 expression using siRNA. Transfection of IEC-18 cells with p38 siRNA for 3 days resulted in over 90% reduction in its expression with minimal overall toxicity when compared with control siRNA-transfected cells (Fig. 6C). When cells were treated with ZD1839, significant phosphorylation of p38 occurred in the control siRNA-transfected group, whereas in p38-silenced cells minimal phosphorylation was detected. Although expressions of Bax and total caspase-3 were unchanged by either siRNA transfection or ZD1839 treatment, the EGFR inhibitor-induced Bax activation and caspase-3 cleavage were reduced significantly in the absence of p38 phosphorylation. Additionally, ZD1839-induced increase in intestinal epithelial cell apoptosis was also significantly suppressed in the background of attenuated p38 expression (Fig. 6D).
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DISCUSSION
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Similar to what we have observed using a mouse model of massive intestinal resection in vivo, we have verified in the present study that EGFR signaling plays an important regulatory role for intestinal epithelial cell apoptosis. Suppression of EGFR activity in IEC-18 cells by either siRNA targeting of EGFR expression or pharmacological use of two different inhibitors resulted in significantly enhanced apoptosis and trafficking of activated Bax to the mitochondria. The magnitude of apoptosis directly correlated with EGFR inhibitor concentration and the extent of EGFR inhibition. On the other hand, when EGFR activity was enhanced by ligand-dependent stimulation, apoptosis was decreased to below baseline. We also showed that Bax expression is required for the apoptotic response to EGFR inhibition. Finally, we revealed that, in the context of EGFR inhibition, p38 MAPK is activated and is necessary for the activation and migration of Bax to the mitochondria to induce apoptosis. Collectively, these data endorse an important role for EGFR signaling to regulate intestinal epithelial cell apoptosis via a pathway of p38 MAPK-dependent activation of Bax.
Although EGFR stimulation generally leads to activation of various downstream signal transduction pathways to include phosphatidylinositol 3-kinase (PI3K), signal transducer and activator of transcription (STAT), and MAPK, EGFR inhibition would be predicted to attenuate downstream signaling pathways. Indeed, we demonstrated EGFR inhibition resulted in impaired phosphorylation of PI3K/Akt, STAT-3, and ERK1/2 (data not shown). However, phosphorylation of p38, but not JNK, was significantly enhanced following EGFR inhibition in our cell line. Conversely, p38 activity was significantly reduced when EGFR was activated, indicating that EGFR activity paradoxically affected the p38 pathway. Although it is generally believed that phosphorylation of p38 is mediated by upstream kinase MAP kinase kinase (MKK) 3/6 in response to variety of stresses (8), we found that MKK3/6 phosphorylation status in response to EGFR modulation remained unchanged (data not shown). Consequently, we believe that phosphorylation of p38 in response to EGFR inhibition was independent of MKK. Interestingly, it has been demonstrated that p38 may undergo autophosphorylation, independent of MKK (9). Another potential link between EGFR inhibition and p38 phosphorylation has been delineated recently in cancer cell lines, suggesting that CARP-1, a novel inducer of apoptosis, played a role in induction of p38 phosphorylation following EGFR inhibition (19). Further studies are needed to understand the precise mechanism by which EGFR activity modulates p38 phosphorylation in intestinal epithelial cells.
With regard to the induction of apoptosis in intestinal epithelial cells, a more established cell survival pathway is mediated by the action of Akt under the regulation of PI3K. In this paradigm, Akt phosphorylates Bax, thus prohibiting its conformational change and mitochondrial translocation to facilitate cell survival (7). Since the PI3K/Akt pathway is directly regulated by the EGFR, we also investigated the role Akt played in the apoptotic response of intestinal epithelial cells in response to EGFR modulation. Although EGFR stimulation resulted in sustained Akt phosphorylation, EGFR inhibition suppressed Akt phosphorylation only briefly (less than 30 min; data not shown). Since significant Bax conformational changes were not detected until 2 h after EGFR inhibition and sustained p38 phosphorylation occurred slightly earlier, it can be inferred that PI3K/Akt pathway probably played a minor role in the induction of apoptosis following EGFR inhibition.
Although p38 MAPK has four isoforms, we illustrated that p38 phosphorylation and expression were required for Bax conformational changes, its mitochondrial translocation, and subsequent induction of apoptosis following EGFR inhibition. The mechanism by which p38 induces Bax activation is presently unknown. Most recently, Bax activation has been demonstrated in hepatoma cell lines via p38-dependent phosphorylation (16). However, it is still unclear whether p38 and Bax can directly interact with each other, because we have failed to coimmunoprecipitate p38 and Bax together. Although other studies have suggested potential links between p38 and Bax activation in response to ionizing radiation (4) or agents that induce the production of reactive oxygen species (10), we believe that this is the first observation that EGFR modulation regulates apoptosis through p38-dependent activation of Bax in nontransformed intestinal epithelial cells. Although the importance of p38 in resection-induced apoptosis is strongly suggested by its significant phosphorylation after SBR, additional studies are being conducted in p38-null mice to further characterize its role in vivo. Through these findings, we propose a novel mechanism for small intestinal homeostasis in which EGFR activity plays a crucial role in the balance between survival and death of enterocytes via regulation of p38 phosphorylation and subsequent conformational changes in Bax. This mechanism opens the possibility for novel therapeutic options in which pharmacological modulation of EGFR or p38 to attenuate apoptosis could enhance the adaptive response of small intestine. Along these lines, we have already demonstrated that combined therapy to attenuate apoptosis (in Bax-deficient mice) as well as to stimulate enterocyte proliferation and attenuate apoptosis further (by EGF treatment) results in a magnified adaptation response to massive SBR (2). Translation of these data into humans could possibly result in significant clinical improvement by regaining valuable absorptive surface area following the loss of large sum of small intestinal length.
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FOOTNOTES
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Address for reprint requests and other correspondence: B. W. Warner, Division of Pediatric Surgery, St. Louis Children's Hospital, One Children's Place, Suite S560, St. Louis, MO 63110 (e-mail: Brad.Warner{at}wustl.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.
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REFERENCES
|
|---|
- Bernal NP, Stehr W, Coyle R, Erwin CR, Warner BW. EGF receptor signaling regulates Bax and Bcl-w expression and apoptotic responses during intestinal adaptation in mice. Gastroenterology 130: 412–423, 2006.[CrossRef][Web of Science][Medline]
- Bernal NP, Stehr W, Profitt S, Erwin CR, Warner BW. Combined pharmacotherapy that increases proliferation and decreases apoptosis optimally enhances intestinal adaptation. J Pediatr Surg 41: 719–724, 2006.[CrossRef][Web of Science][Medline]
- Chen Z, Gibson TB, Robinson F, Silvestro L, Pearson G, Xu B, Wright A, Vanderbilt C, Cobb MH. MAP kinases. Chem Rev 101: 2449–2476, 2001.[CrossRef][Web of Science][Medline]
- Choi SY, Kim MJ, Kang CM, Bae S, Cho CK, Soh JW, Kim JH, Kang S, Chung HY, Lee YS, Lee SJ. Activation of Bak and Bax through c-abl-protein kinase Cdelta-p38 MAPK signaling in response to ionizing radiation in human non-small cell lung cancer cells. J Biol Chem 281: 7049–7059, 2006.[Abstract/Free Full Text]
- Evers BM, Chung DH, Townsend CM Jr, Thompson JC. Molecular mechanisms of intestinal adaptation after resection. Am Coll Surg Surg Forum 42: 130–132, 1991.
- Frey MR, Dise RS, Edelblum KL, Polk DB. p38 Kinase regulates epidermal growth factor receptor downregulation and cellular migration. EMBO J 25: 5683–5692, 2006.[CrossRef][Web of Science][Medline]
- Gardai SJ, Hildeman DA, Frankel SK, Whitlock BB, Frasch SC, Borregaard N, Marrack P, Bratton DL, Henson PM. Phosphorylation of Bax Ser184 by Akt regulates its activity and apoptosis in neutrophils. J Biol Chem 279: 21085–21095, 2004.[Abstract/Free Full Text]
- Garrington TP, Johnson GL. Organization and regulation of mitogen-activated protein kinase signaling pathways. Curr Opin Cell Biol 11: 211–218, 1999.[CrossRef][Web of Science][Medline]
- Ge B, Gram H, Di PF, Huang B, New L, Ulevitch RJ, Luo Y, Han J. MAPKK-independent activation of p38alpha mediated by TAB1-dependent autophosphorylation of p38alpha. Science 295: 1291–1294, 2002.[Abstract/Free Full Text]
- Gomez-Lazaro M, Galindo MF, Melero-Fernandez de Mera RM, Fernandez-Gomez FJ, Concannon CG, Segura MF, Comella JX, Prehn JH, Jordan J. Reactive oxygen species and p38 mitogen-activated protein kinase activate Bax to induce mitochondrial cytochrome c release and apoptosis in response to malonate. Mol Pharmacol 71: 736–743, 2007.[Abstract/Free Full Text]
- Hackel PO, Zwick E, Prenzel N, Ullrich A. Epidermal growth factor receptors: critical mediators of multiple receptor pathways. Curr Opin Cell Biol 11: 184–189, 1999.[CrossRef][Web of Science][Medline]
- Haxhija EQ, Yang H, Spencer AU, Sun X, Teitelbaum DH. Influence of the site of small bowel resection on intestinal epithelial cell apoptosis. Pediatr Surg Int 22: 37–42, 2006.[CrossRef][Web of Science][Medline]
- Helmrath MA, Erwin CR, Shin CE, Warner BW. Enterocyte apoptosis is increased following small bowel resection. J Gastrointest Surg 2: 44–49, 1998.[CrossRef][Medline]
- Helmrath MA, Shin CE, Erwin CR, Warner BW. The EGF/EGF-receptor axis modulates enterocyte apoptosis during intestinal adaptation. J Surg Res 77: 17–22, 1998.[CrossRef][Web of Science][Medline]
- Jarboe MD, Juno RJ, Bernal NP, Knott AW, Zhang Y, Erwin CR, Warner BW. Bax deficiency rescues resection-induced enterocyte apoptosis in mice with perturbed EGF receptor function. Surgery 136: 121–126, 2004.[CrossRef][Web of Science][Medline]
- Kim BJ, Ryu SW, Song BJ. JNK- and p38 kinase-mediated phosphorylation of Bax leads to its activation and mitochondrial translocation and to apoptosis of human hepatoma HepG2 cells. J Biol Chem 281: 21256–21265, 2006.[Abstract/Free Full Text]
- O'Brien DP, Nelson LA, Huang FS, Warner BW. Intestinal adaptation: structure, function, regulation. Semin Pediatr Surg 10: 56–64, 2001.[CrossRef][Medline]
- Panis Y, Lomri N, Emond JC. Early gene expression associated with regeneration is intact after massive hepatectomy in rats. J Surg Res 79: 103–108, 1998.[CrossRef][Web of Science][Medline]
- Rishi AK, Zhang L, Yu Y, Jiang Y, Nautiyal J, Wali A, Fontana JA, Levi E, Majumdar AP. Cell cycle- and apoptosis-regulatory protein-1 is involved in apoptosis signaling by epidermal growth factor receptor. J Biol Chem 281: 13188–13198, 2006.[Abstract/Free Full Text]
- Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell 103: 211–225, 2000.[CrossRef][Web of Science][Medline]
- Stern LE, Huang F, Kemp CJ, Falcone RA Jr, Erwin CR, Warner BW. Bax is required for increased enterocyte apoptosis after massive small bowel resection. Surgery 128: 165–170, 2000.[CrossRef][Web of Science][Medline]
- Tang Y, Swartz-Basile DA, Swietlicki EA, Yi L, Rubin DC, Levin MS. Bax is required for resection-induced changes in apoptosis, proliferation, and members of the extrinsic cell death pathways. Gastroenterology 126: 220–230, 2004.[CrossRef][Web of Science][Medline]
- Tang Y, Swietlicki EA, Jiang S, Buhman KK, Davidson NO, Burkly LC, Levin MS, Rubin DC. Increased apoptosis and accelerated epithelial migration following inhibition of hedgehog signaling in adaptive small bowel postresection. Am J Physiol Gastrointest Liver Physiol 290: G1280–G1288, 2006.[Abstract/Free Full Text]
- Thompson JS, Barent B. Effects of intestinal resection on enterocyte apoptosis. J Gastrointest Surg 3: 672–677, 1999.[CrossRef][Web of Science][Medline]
- Van Laethem A, Van Kelst S, Lippens S, Declercq W, Vandenabeele P, Janssens S, Vandenheede JR, Garmyn M, Agostinis P. Activation of p38 MAPK is required for Bax translocation to mitochondria, cytochrome c release and apoptosis induced by UVB irradiation in human keratinocytes. FASEB J 18: 1946–1948, 2004.[Abstract/Free Full Text]
- Warner BW, Erwin CR. Critical roles for EGF receptor signaling during resection-induced intestinal adaptation. J Pediatr Gastroenterol Nutr 43, Suppl 1: S68–S73, 2006.[CrossRef][Web of Science][Medline]
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Copyright © 2007 by the American Physiological Society.
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ABSTRACT
MATERIALS AND METHODS
