Epidermal Growth Factor Receptor–Mediated Proliferation of Enterocytes Requires p21waf1/cip1 Expression
2006; Elsevier BV; Volume: 131; Issue: 1 Linguagem: Inglês
10.1053/j.gastro.2006.05.007
ISSN1528-0012
AutoresGeorge Sheng, Kathryn Q. Bernabe, Jun Guo, Brad W. Warner,
Tópico(s)Colorectal Cancer Treatments and Studies
ResumoBackground & Aims: Epidermal growth factor receptor (EGFR)-mediated increase in enterocyte proliferation following massive resection is a major mechanism by which the small intestine adapts to the loss of its mucosal surface area. In addition, expression of the cyclin-dependent kinase inhibitor p21waf1/cip1 is required for resection-induced enterocyte proliferation. This study sought to establish a mechanistic link between EGFR-mediated intestinal epithelial cell proliferation and p21waf1/cip1 expression. Methods: EGF was used to stimulate IEC-6 and HCA-7 cells. P21waf1/cip1 messenger RNA (mRNA) and protein expression were measured by real-time polymerase chain reaction and Western blot, respectively. P21waf1/cip1 promoter studies were performed using p21waf1/cip1 promoter-driven luciferase assay. Pharmacologic inhibitors of PI3-kinase and mitogen activated protein kinase (MAPK) were used to block these pathways downstream of the activated EGFR. Constitutively active Ras, Raf, or MEK-1 constructs were transfected into cells for overexpression studies. Cell proliferation was measured by bromodeoxyuridine incorporation following p21waf1/cip1 silencing with RNAi. Finally, Cyclin D1/Cdk interaction was evaluated by immunoprecipitation. Results: EGFR activation in intestinal epithelial cells induced the expression of p21waf1/cip1 mRNA and protein This event was transcriptionally regulated via a 50-bp segment of the p21waf1/cip1 promoter as a result of MAPK activation. Exogenous EGF failed to induce proliferation in p21waf1/cip1-silenced cells and adaptive proliferation after intestinal resection in p21waf1/cip1-null mice. Functionally, p21waf1/cip1 up-regulation was required for stabilizing Cyclin D/Cdk 4 complexes and intestinal cell proliferation. Conclusions: EGFR-mediated induction of enterocyte proliferation requires MAPK-dependent increase in p21waf1/cip1 expression in intestinal epithelial cells. These studies elucidate an important mechanism for resection-induced enterocyte proliferation during intestinal adaptation. Background & Aims: Epidermal growth factor receptor (EGFR)-mediated increase in enterocyte proliferation following massive resection is a major mechanism by which the small intestine adapts to the loss of its mucosal surface area. In addition, expression of the cyclin-dependent kinase inhibitor p21waf1/cip1 is required for resection-induced enterocyte proliferation. This study sought to establish a mechanistic link between EGFR-mediated intestinal epithelial cell proliferation and p21waf1/cip1 expression. Methods: EGF was used to stimulate IEC-6 and HCA-7 cells. P21waf1/cip1 messenger RNA (mRNA) and protein expression were measured by real-time polymerase chain reaction and Western blot, respectively. P21waf1/cip1 promoter studies were performed using p21waf1/cip1 promoter-driven luciferase assay. Pharmacologic inhibitors of PI3-kinase and mitogen activated protein kinase (MAPK) were used to block these pathways downstream of the activated EGFR. Constitutively active Ras, Raf, or MEK-1 constructs were transfected into cells for overexpression studies. Cell proliferation was measured by bromodeoxyuridine incorporation following p21waf1/cip1 silencing with RNAi. Finally, Cyclin D1/Cdk interaction was evaluated by immunoprecipitation. Results: EGFR activation in intestinal epithelial cells induced the expression of p21waf1/cip1 mRNA and protein This event was transcriptionally regulated via a 50-bp segment of the p21waf1/cip1 promoter as a result of MAPK activation. Exogenous EGF failed to induce proliferation in p21waf1/cip1-silenced cells and adaptive proliferation after intestinal resection in p21waf1/cip1-null mice. Functionally, p21waf1/cip1 up-regulation was required for stabilizing Cyclin D/Cdk 4 complexes and intestinal cell proliferation. Conclusions: EGFR-mediated induction of enterocyte proliferation requires MAPK-dependent increase in p21waf1/cip1 expression in intestinal epithelial cells. These studies elucidate an important mechanism for resection-induced enterocyte proliferation during intestinal adaptation. The epithelium of the small intestine undergoes rapid and continuous renewal whereby loss of differentiated enterocytes from the villus tips is matched by a continuous rate of enterocyte production within the crypts. One of the most potent stimuli for enterocyte proliferation is a massive small bowel resection (SBR) during a compensatory process referred to as adaptation.1Williamson R.C. Intestinal adaptation Structural, functional and cytokinetic changes.N Engl J Med. 1978; 298: 1393-1402Crossref PubMed Scopus (487) Google Scholar, 2Urban E. Weser E. Intestinal adaptation to bowel resection.Adv Int Med. 1980; 26: 265-291PubMed Google Scholar, 3O'Brien D.P. Nelson L.A. Huang F.S. Warner B.W. Intestinal adaptation structure, function, and regulation.Semin Pediatr Surg. 2001; 10: 56-64Abstract Full Text PDF PubMed Scopus (108) Google Scholar During resection-induced adaptation, increased rates of enterocyte proliferation contribute toward the generation of taller villi and deeper crypts, thus amplifying the absorptive and digestive mucosal surface area of the remnant gut. Illumination of the mechanism and mediators of adaptation is fundamental to the design of therapy intended to enhance maximally this response in patients with the short gut syndrome, thereby encouraging independence from parenteral nutrition. Past research in our laboratory has established the importance of intact epidermal growth factor receptor (EGFR) signaling in the pathogenesis of normal intestinal adaptation. Experimental paradigms in which the EGFR is either stimulated or inhibited after massive SBR results in an amplified or attenuated adaptation response, respectively.4Stern L.E. Erwin C.R. O'Brien D.P. Huang F. Warner B.W. Epidermal growth factor is critical for intestinal adaptation following small bowel resection.Microsc Res Tech. 2000; 51: 138-148Crossref PubMed Scopus (59) Google Scholar In additional experiments using laser capture microdissection microscopy, we have observed that expression of the Cyclin-dependent kinase inhibitor (CDKI) p21waf1/cip1 is elevated in the region of proliferating crypt enterocytes in association with increased enterocyte proliferation and adaptation.5Stehr W. Bernal N.P. Erwin C.R. Bernabe K.Q. Guo J. Warner B.W. Roles for p21waf1/cip1 and p27kip1 during the adaptation response to massive intestinal resection.Am J Physiol Gastrointest Liver Physiol. 2006; 290: G933-G941Crossref PubMed Scopus (13) Google Scholar In accordance with increased intestinal expression of p21waf1/cip1 during resection-induced proliferation, mice deficient in p21waf1/cip1 expression failed to exhibit increases in rates of enterocyte proliferation and other parameters of adaptation such as ileal wet weight, villus height, and crypt depth following a 50% SBR.6Stern L.E. Falcone Jr, R.A. Kemp C.J. Erwin C.R. Warner B.W. p21 (WAF1/CIP1) is required for the mitogenic response to intestinal resection.J Surg Res. 2000; 90: 45-50Abstract Full Text PDF PubMed Scopus (23) Google Scholar Although predominately considered to be a cell-cycle inhibitor, p21waf1/cip1 has multiple other functions, depending on the cell type and stimulus. At low expression levels, p21waf1/cip1 is believed to assist in the assembly of Cyclin/Cdk complexes, thus promoting cell-cycle entry and proliferation.7Cheng M. Olivier P. Diehl J.A. Fero M. Roussel M.F. Roberts J.M. Sherr C.J. The p21(Cip1) and p27(Kip1) CDK 'inhibitors' are essential activators of Cyclin D-dependent kinases in murine fibroblasts.EMBO J. 1999; 18: 1571-1583Crossref PubMed Scopus (990) Google Scholar On the other hand, when p21waf1/cip1 protein is highly abundant, the catalytic activity of Cyclin/Cdk complexes is inhibited, and cell-cycle arrest occurs.8Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 Cyclin-dependent kinases.Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5442) Google Scholar, 9Wilson J.W. Pritchard D.M. Hickman J.A. Potten C.S. Radiation-induced p53 and p21WAF-1/CIP1 expression in the murine intestinal epithelium apoptosis and cell cycle arrest.Am J Pathol. 1998; 153: 899-909Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 10Gartel A.L. Tyner A.L. Transcriptional regulation of the p21(WAF1/CIP1) gene.Exp Cell Res. 1999; 246: 280-289Crossref PubMed Scopus (585) Google Scholar In addition, through the activation of mitogen-activated protein kinase (MAPK), p21waf1/cip1 has also been associated with the processes of apoptosis and differentiation.11Woods D. Parry D. Cherwinski H. Bosch E. Lees E. McMahon M. Raf-induced proliferation or cell cycle arrest is determined by the level of Raf activity with arrest mediated by p21Cip1.Mol Cell Biol. 1997; 17: 5598-5611Crossref PubMed Scopus (583) Google Scholar At the level of transcription, p21waf1/cip1 is regulated through both p53-dependent and p53-independent pathways. In the field of colon cancer research, p53-dependent transcriptional regulation of p21waf1/cip1 and its function as a cell-cycle inhibitor have been extensively studied. In various other cell lines, the induction of p21waf1/cip1 expression occurs independently of p53 through interaction of various transcription factors with cis-acting elements located within the p21waf1/cip1 promoter.10Gartel A.L. Tyner A.L. Transcriptional regulation of the p21(WAF1/CIP1) gene.Exp Cell Res. 1999; 246: 280-289Crossref PubMed Scopus (585) Google Scholar However, the regulation of p21waf1/cip1 expression in the small intestine and its role in enterocyte proliferation remains poorly characterized. Because both EGFR signaling and p21waf1/cip1 expression are necessary for enterocyte proliferation and adaptation in response to SBR, the present study was designed to elucidate the mechanism by which EGFR regulates p21waf1/cip1 expression. In the current study, we used both a nontransformed rat small intestine cell line (IEC-6), as well as a transformed human colon cancer cell line (HCA-7) in separate but complementary studies to study the relationship between EGFR-induced enterocyte proliferation and p21waf1/cip1 expression. We report for the first time that p21waf1/cip1 expression is required for the proliferative response of intestinal epithelial cells to EGF. Furthermore, we reveal an important mechanistic link between the activated EGFR and p21waf1/cip1 expression via the MAPK pathway. The protocol for this study was approved by the Children's Hospital Research Foundation Institutional Animal Care and Use Committee (Cincinnati Children's Hospital Medical Center, Cincinnati, OH). In the first series of experiments, a time course for EGFR activation following EGF treatment of intestinal epithelial cell lines was determined. Using this information, p21waf1/cip1 messenger RNA (mRNA) and protein expression were determined in response to EGFR stimulation and inhibition. In the next set of experiments, the region of the p21waf1/cip1 promoter activated by EGFR signaling was determined using a luciferase reporter ligated to a series of truncated p21waf1/cip1 promoter constructs, which were transfected into the cells. Next, the effect of inhibiting several candidate signaling pathways downstream of the EGFR on p21waf1/cip1 promoter activity and protein expression were determined. Alternatively, transient overexpression of Ras, Raf, and MEK1 on p21waf1/cip1 promoter activity and protein expression were measured. Finally, RNA interference (RNAi) was used to determine the effect of silenced p21waf1/cip1 expression on EGF-directed intestinal epithelial cell proliferation, as well as expression and interactions with other cell-cycle regulatory proteins. The HCA-7 cell line was a gift of Dr Susan Kirkland (University of London, London, United Kingdom) and maintained in McCoy's 5A modified medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. IEC-6 cells from ATCC were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mmol/L glutamine. The PI3-kinase inhibitor LY 294002 and the MAPK inhibitor UO126 were purchased from Cell Signaling (Beverly, MA). Monoclonal anti-Cdk4 antibody and anti-p21waf1/cip1 antibody were 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. Monoclonal Cyclin D1 and polyclonal ERK1/2 antibodies were from Cell Signaling. For direct Western blot, parental or transfected HCA-7 and IEC-6 cells were lysed in 1X SDS sample buffer (50 mmol/L Tris HCL, pH 6.8, 2% SDS, 10% glycerol, and 5% mercaptoethanol). For immunoprecipitation, the cells were washed with ice cold phosphate-buffered saline (PBS) and then lysed for 30 minutes in lysis buffer containing 50 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl, 5 mmol/L EDTA, 1% Triton X-100 plus protease inhibitor tablet (Roche, Indianapolis, IN) and phosphatase inhibitor cocktail I/II (Sigma Chemical Co, St. Louis, MO). The lysates were then centrifuged at 20,000g for 30 minutes at 4°C. The supernatant was then incubated with protein A plus G beads (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hour (preclearing) at 4°C. Following this, the beads were spun down and discarded, and the supernatant was further incubated with protein A plus G beads and appropriate antibody overnight at 4°C. After washing in lysis buffer, the absorbed complexes were removed from the beads by heating for 5 minutes 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. RNA was isolated with RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer. Briefly, cells were lysed in a culture dish, lysates were run through RNeasy column, and RNA was eluted and collected in RNase-free water. The isolated RNA was converted to complementary DNA (cDNA) using a conventional reverse-transcriptase (RT) reaction (GIBCO BRL, Gaithersberg, MD). The cDNA concentration was then quantified using OligoGreen (Molecular Probes, Eugene, OR) and a Fluorometer (Turner Deign, Sunnyvale, CA) following the instructions of the manufacturers. For quantification of p21waf1/cip1 mRNA expression, 100 ng cDNA was loaded and real-time polymerase chain reaction (PCR) performed according to the protocol from the SuperArray Gene Expression Assay Kit (SuperArray Bioscience, Frederick, MD). In brief, real-time PCR was done using a Smart Cycler System (Cepheid, Sunnyvale, CA) and the LightCycler RNA Master SYBR Green I kit (Roche Diagnostics AG, Rotkreuz, Switzerland) with SYBR Green I dye at concentrations optimized by the manufacturer. Each set of oligonucleotides used for real-time PCR was designed to produce an amplicon of approximately 200 base pair (bp) and was optimized for primer, MgCl2 concentration, annealing temperature, and cycle times. Generally, 45 cycles were done, and SYBR Green fluorescence was measured after each elongation step. At the end of the PCR, a melting curve analysis was performed by gradually increasing the temperature from 60°C to 95°C (0.1°C/s) to determine purity. After the PCR was completed, the SYBR Green fluorescent signal was compared to produce a relative fold increase in copies of target molecules compared with control. The p21waf1/cip1 promoter-luciferase reporter constructs p21P, p21PΔ53, p21PΔ1.1, p21PΔ1.9, p21Psma, p21PsmaΔ1, and p21PsmaΔ2 were generously provided by Dr Xiao-Fan Wang (Department of Pharmacology, Duke University Medical Center, Durham, NC). IEC-6 or HCA-7 cells were plated in 24-well plates for 24 hours. The resulting monolayer (80% confluence) was washed with serum-free medium before transient cotransfection with 0.5 μg p21waf1/cip1 promoter-luciferase plasmid or 0.1 μg renilla luciferase expression plasmid, pRL-SV40 (Promega, Madison, WI) using a Fugene 6 transfection kit (Roche, Indianapolis, IN). Twenty-four hours after transfection, the medium was replaced with serum-free DMEM, and cells were incubated for another 24 hours for G0 synchronization. Following EGF stimulation, cells were washed, collected, and extracted in 100 μL Passive Lysis Buffer (Promega) before analysis of firefly luciferase activity using the Dual-Luciferase reporter assay system (Promega). P21waf1/cip1 promoter activity was normalized to the amount of renilla-based luminescence to correct for differences in transfection efficiency. For both IEC-6 and HCA-7 cell lines, 1 × 106 cells were transfected with p21waf1/cip1 siRNA (SMARTpool p21 reagent; Dharmacon, Lafayette, CO) and control siRNA (siCONTROL nontargeting siRNA) (Dharmacon) at 100 nmol/L concentration with X-tremeGENE (Roche, Indianapolis, IN), according to the manufacturer's protocol. Cells were collected 48 hours after transfection and used for experiments. To confirm silencing, cells were collected, and Western blots were performed to measure p21waf1/cip1 protein. Cells were split and grown in 24-well plates to 50% confluence. One day before experimentation, the cells were serum starved for 24 hours and then stimulated with EGF (100 ng/mL) for 16 hours. The cell proliferation study was performed using Cell Proliferation ELISA, BrdU kit (Roche, Indianapolis, IN), according to the manufacturer's instructions. Experiments were carried out in quadruplicate. For comparisons of mean values between the 2 groups, the Student t test was used. For comparisons of mean values among more than 2 groups, a 1-way analysis of variance was used. Statistical significance was established at a P value of <.05. Stimulation of both IEC-6 and HCA-7 cells with EGF induced a rapid (within 15 minutes) activation of the EGFR, which persisted for 1 hour (Figure 1). This was demonstrated by immunoprecipitation of the EGFR and subsequent immunoblotting for phosphotyrosine. Total (nonphosphorylated) EGFR remained constant initially, but began to decrease after EGF stimulation, presumably because of receptor internalization and degradation.12Schlessinger J. Cell signaling by receptor tyrosine kinases.Cell. 2000; 103: 211-225Abstract Full Text Full Text PDF PubMed Scopus (3618) Google Scholar In IEC-6 cells, EGF stimulation resulted in a 3-fold increase in p21waf1/cip1 mRNA by 1 hour (Figure 2A) with a return to baseline after 4 hours. Without EGF, both IEC-6 and HCA-7 cells had low levels of p21waf1/cip1 protein. Following EGFR stimulation, elevated p21waf1/cip1 protein expression was detected by 2 hours (Figure 2B). This increased expression persisted for at least 24 hours after EGF treatment. We verified that serum starvation for up to 48 hours alone did not induce the expression of p21waf1/cip1 protein in these cells (data not shown). Both cell lines appeared to respond to EGF stimulation identically with regard to p21waf1/cip1 expression. Pretreatment of cells with a selective EGFR tyrosine kinase inhibitor (ZD1839; IRESSA; AstraZeneca, Cheshire, England) for 2 hours abolished EGF-induced p21waf1/cip1expression, thus confirming the involvement of EGFR activation in the induction of p21waf1/cip1 expression by EGF stimulation (Figure 2C). The expression of p21waf1/cip1 in response to EGFR activation was then studied at a transcriptional level by employing a p21waf1/cip1 promoter luciferase assay. Both IEC-6 and HCA-7 cells were cotransfected with a 2.4 kilobase (kb) human p21waf1/cip1 promoter-driven luciferase construct and an SV40-directed renilla construct as a control. At 24 hours following transfection, cells were stimulated with EGF, resulting in a 2- and 3-fold increase in p21waf1/cip1 promoter activity in IEC-6 and HCA-7 cells, respectively. One step toward defining the mechanism by which EGFR activity induces the expression of p21waf1/cip1 is to determine the region of the p21waf1/cip1 promoter affected by EGFR signaling. A series of 5′ promoter deletion mutants of the p21waf1/cip1 promoter proximal to the transcriptional initiation site were transfected into HCA-7 cells for 24 hours. Following EGF stimulation, promoter-driven luciferase activity was measured. In the full-length promoter construct p21P, p21waf1/cip1 promoter activity was activated 3-fold by EGF, whereas the effect of EGF on the minimal promoter construct (containing only 62 bp; p21PsmaΔμμ was abolished (Figure 3). When the panel of deletion mutants was assayed, the region of the promoter responsible for induction by EGF was determined to be within a 500-bp region near the transcriptional initiation site. The P21Psma construct that contained the p21waf1/cip1 promoter sequences from base −111 through the transcriptional initiation site was equally activated by EGF stimulation, despite a decrease in baseline promoter activity, thus further targeting the EGF-responsive element to a proximal region of the promoter between the −111 and −61 bp. When this segment was removed from the p21waf1/cip1 full-length promoter (p21PsmaΔ2), EGF-induced luciferase activity was eliminated. These results indicate that activation of p21waf1/cip1 promoter activity in response to EGFR signaling requires the bp sequence between −61 and −111 on the p21waf1/cip1 promoter. To identify a signal transduction pathway involved in EGFR-mediated p21waf1/cip1 expression, pharmacologic inhibitors of MEK1/2 (UO126) and PI3 kinase (LY294002) were used. Both quiescent IEC-6 and HCA-7 cells were pretreated with the inhibitors for 2 hours prior to EGF stimulation, and p21waf1/cip1 protein expression levels were analyzed. Although the PI3 kinase inhibitor LY294002 was able to inhibit completely AKT phosphorylation, it failed to abolish EGFR induction of p21waf1/cip1 protein expression (Figure 4). In the IEC-6 cells, LY294002 treatment was associated with a slightly reduced expression of p21waf1/cip1 in response to EGF, whereas, in the HCA-7 cell line, p21waf1/cip1 protein expression was slightly elevated. On the other hand, the selective Erk1/2 inhibitor UO126 was able to suppress Erk phosphorylation as well as EGFR-mediated p21waf1/cip1 expression (Figure 4). Using the luciferase reporter system, UO126 completely abolished the EGFR-mediated induction of p21waf1/cip1 promoter activity, whereas LY294002 had no effect (Figure 5). These findings would suggest that, in the IEC-6 cells, the PI3K pathway likely affects p21waf1/cip1 protein stability, rather than regulating transcription.Figure 5The effect of PI3-K or MAPK inhibition on p21 promoter activity in IEC-6 cells treated with EGF. Cells were cotransfected with either the 2.4-kb full-length human P21waf1/cip1 promoter or SV-40 promoter-driven renilla luciferase plasmids. Following transfection, cells were pretreated with either the PI-K inhibitor LY294002 or the MAPK inhibitor UO126 and then stimulated for 1 hour with EGF (100 ng/mL). The resulting luciferase activity was compared with cells without EGF treatment. The graph represents fold differences in luciferase activity relative to unstimulated cells. Experiments were performed in quadruplicate for each time point; *denotes P < .05 vs unstimulated cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To validate further that p21waf1/cip1 expression is regulated by the MAPK pathway, quiescent HCA-7 cells were transfected with constitutively active Ras, Raf, or MEK1 constructs. Activation of the MAPK pathway was verified as a result of increased ERK phosphorylation without affecting total ERK expression (Figure 6A). In association with the activation of ERK by each of the MAPK transfection constructs, the expression of p21waf1/cip1 protein was also elevated. Correspondingly, p21waf1/cip1 promoter activity following transfection with constitutively active Ras, Raf, or MEK1 was also significantly elevated (Figure 6B). To address the functional significance of p21waf1/cip1 in response to EGFR stimulation, p21waf1/cip1 expression was silenced using siRNA in both IEC-6 and HCA-7 cells. Overall, p21waf1/cip1 expression silencing efficiency was approximately 80% in both cell lines and was not associated with significant toxicity as revealed by increases in apoptosis (Figure 7) or changes in cell morphology. After transfection with p21waf1/cip1 siRNA for 48 hours, both cell lines exhibited a significant decrease in both baseline and EGF-induced p21waf1/cip1 expression (Figure 8). After EGF stimulation for 24 hours, IEC-6 cells transfected with control RNAi showed a 40% increase in proliferation. In contrast, cells that were transfected with p21waf1/cip1 siRNA did not proliferate in response to EGF. This was verified by lack of change in cell number at the end of the experiment. Similar to our findings in IEC-6 cells, HCA-7 cells transfected with control RNAi exhibited greater than 60% increase in proliferation after EGF treatment, whereas p21waf1/cip1-silenced cells had a minimal proliferation response to EGF (Figure 8). In contrast, blocking p21waf1/cip1 expression in the context of serum stimulation only slightly reduced cell proliferation (data not shown). Thus, the effect of preventing p21waf1/cip1 expression in intestinal epithelial cells was fairly selective for EGFR-stimulated proliferation.Figure 8Effect of EGF stimulation of proliferation and p21waf1/cip1 protein expression in IEC-6 and HCA-7 cells following p21waf1/cip1 RNA silencing. P21 siRNA and siControl (scrambled) constructs were transfected into cells. After confirmation of attenuated p21waf1/cip1 expression by Western blotting, cells were stimulated with EGF (100 ng/mL), and proliferation was measured using a BrdU ELISA Assay Kit (Roche). A proliferation index was derived as the percentage of proliferating cells; *denotes P < .05 EGF-stimulated vs nonstimulated cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To study further the mechanism for attenuated expression of p21waf1/cip1 expression for the inhibition of intestinal epithelial cell proliferation following EGFR activation, Cyclin D1 and Cdk4 association after p21waf1/cip1 silencing was evaluated. As demonstrated in Figure 9, the expression of Cdk4 remained constant, despite either EGF stimulation or p21waf1/cip1 silencing. In contrast, the expected increase in Cyclin D1 expression in response to EGF was seen. This pattern was unaffected by preventing p21waf1/cip1 expression using siRNA. When Cdk4 was immunoprecipitated, the expected association of p21waf1/cip1 protein with Cdk4 was diminished in cells transfected with p21waf1/cip1 siRNA (Figure 10). Furthermore, Cyclin D1 association with Cdk4 was also significantly attenuated when p21waf1/cip1 expression was silenced (Figure 10). These results suggest that the interaction between Cdk4 and Cyclin D1 as well as proliferation are dramatically reduced when p21waf1/cip1 expression is suppressed. To correlate our in vitro findings of the critical relationship between EGFR signaling and p21waf1/cip1 with what takes place in vivo, we first measured the intestinal expression of p21waf1/cip1 in wild-type (WT; C57/Bl6) control mice as well as in mice with genetically modified EGFR-signaling capacity (Figure 11A). p21waf1/cip1 Protein expression was measured in mucosal scrapings of ileum as we have previously described.13Stehr W. Mercer T.I. Bernal N.P. Erwin C.R. Warner B.W. Opposing roles for p21waf1/cip1 and p27kip1 in enterocyte differentiation, proliferation, and migration.Surgery. 2005; 138: 187-194Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar When compared with the WT mice, transgenic mice in which EGF is overexpressed in the intestine had elevated levels (by approximately 40%) of p21waf1/cip1. These mice were created by our laboratory and demonstrate an amplified adaptation response to intestinal resection.14Erwin C.R. Helmrath M.A. Shin C.E. Falcone Jr, R.A. Stern L.E. Warner B.W. Intestinal overexpression of EGF in transgenic mice enhances adaptation after small bowel resection.Am J Physiol. 1999; 277: G533-G540PubMed Google Scholar Alternatively, intestinal levels of p21waf1/cip1 were approximately 50% lower in wave-2 mice that have diminished EGFR signaling capacity.15Luetteke N.C. Phillips H.K. Qiu T.H. Copeland N.G. Earp H.S. Jenkins N.A. Lee D.C. The mouse waved-2 phenotype results from a point mutation in the EGF receptor tyrosine kinase.Genes Dev. 1994; 8: 399-413Crossref PubMed Scopus (401) Google Scholar, 16Fowler K.J. Walker F. Alexander W. Hibbs M.L. Nice E.C. Bohmer R.M. Mann G.B. Thumwood C. Maglitto R. Danks J.A. et al.A mutation in the epidermal growth factor receptor in waved-2 mice has a profound effect on receptor biochemistry that results in impaired lactation.Proc Natl Acad Sci U S A. 1995; 92: 1465-1469Crossref PubMed Scopus (189) Google Scholar We have previously revealed that these mice have markedly impaired proliferation and adaptation responses to intestinal resection.17Helmrath M.A. Erwin C.R. Warner B.W. A defective EGF receptor in waved-2 mice attenuates intestinal adaptation.J Surg Res. 1997; 69: 76-80Abstract Full Text PDF PubMed Scopus (93) Google Scholar Finally, we determined whether the impaired proli
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