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Role of eIF3a (eIF3 p170) in intestinal cell differentiation and its association with early development
r 2007, Copyright the Authors Differentiation (2007) 75:652–661 DOI: 10.1111/j.1432-0436.2007.00165.x Journal compilation r 2007, International Society of Differentiation
OR IGI N A L A R T IC L E
Zhaoqian Liu . Zizheng Dong . Zuocheng Yang . Qun Chen . Yi Pan . Youyun Yang . Ping Cui . Xin Zhang . Jian-Ting Zhang
Role of eIF3a (eIF3 p170) in intestinal cell differentiation and its association with early development
Received July 21, 2006; accepted in revised form December 21, 2006
Abstract Eukaryotic initiation factor 3a (eIF3a) has been suggested to play a regulatory role in mRNA translation. Decreased eIF3a expression has been observed in differentiated cells while higher levels have been observed in cancer cells. However, whether eIF3a plays any role in differentiation and development is currently unknown. Here, we investigated eIF3a expression during mouse development and its role in differentiation of colon epithelial cells. We found that eIF3a expression was higher in fetal tissues compared with postnatal ones. Its expression in intestine, stomach, and
lung abruptly stopped on the 18th day in gestation but persisted in liver, kidney, and heart throughout the postnatal stage at decreased levels. Similarly, eIF3a expression in colon cancer cell lines, HT-29 and Caco-2, drastically decreased prior to differentiation. Enforced eIF3a expression inhibited while knocking it down using small interference RNA promoted Caco-2 differentiation. Thus, eIF3a may play some roles in development and differentiation and that the decreased eIF3a expression may be a pre-requisite of intestinal epithelial cell differentiation.
Zhaoqian Liu 1,2 Zizheng Dong1 Qun Chen Youyun . ) Yang Ping Cui Jian-Ting Zhang (* Department of Pharmacology and Toxicology Walther Oncology Center Walther Cancer Institute Indiana University School of Medicine 1044 W. Walnut Street Indianapolis, IN 46202, U.S.A. Tel: 11 317 278 4503 Fax: 11 317 274 8046 E-mail: [email protected]
Key words translational regulation protein synthesis initiation factor cancer Caco-2
Zuocheng Yang1 Department of Pediatrics The Third Xiangya Hospital Central South University Changsha, Hunan 410013, China Yi Pan Xin Zhang Department of Medical and Molecular Genetics Indiana University School of Medicine 975 W. Walnut Street Indianapolis, IN 46202, U.S.A. 1
These authors contributed equally.
2 Present address: Institute of Clinical Pharmacology, Xiangya Medical School, Central South University, Changsha, Hunan 410078, China.
Introduction The initiation step in eukaryotic protein synthesis is generally promoted by eukaryotic initiation factors (eIFs) (Dong and Zhang, 2006). Among these factors, eIF3 is the largest complex consisting of 13 subunits and it plays an essential role in translation by binding directly to the 40S ribosomal subunit and promoting formation of the 40S pre-initiation complex (Dong and Zhang, 2006). However, one of the 13 subunits of eIF3, eIF3a (p170), has been thought not to be required for the function of eIF3 in translation initiation (Chaudhuri et al., 1997) and it may play some role as a regulator for translation of a subset of mRNAs (Dong and Zhang, 2003, 2006; Dong et al., 2004). It has been found that the expression of eIF3a is elevated in human cancers of lung (Pincheira et al., 2001b), breast (Bachmann et al., 1997), esophagus (Chen and Burger, 1999), cervix (Dellas et al., 1998), and stomach (Chen and Burger, 2004). It has also been found that the expression of eIF3a is decreased in differentiated cells in culture (Bachmann
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et al., 1997). While we have shown that eIF3a expression plays some roles in cell proliferation and carcinogenesis and suppressing eIF3a expression could reverse the malignant phenotype of lung and breast cancer cells (Dong et al., 2004), it remains unknown whether eIF3a plays any role in cell differentiation and development. The gastrointestinal tract represents one of the best models for studying mechanisms of development and differentiation. In mouse, the intestinal tube forms at approximately 8–10 days of gestation (Kedinger, 1994). The digestive organs including liver, stomach, duodenum, and pancreas originate from the embryonic gut. The mammalian intestinal mucosa generally undergoes a process of continued renewal characterized by active proliferation of stem cells localized near the base of the crypts, progression of these cells up the crypt-villas with cessation of proliferation, and subsequent differentiation into one of the four primary cell types including goblet, enterocytes, paneth, and enteroendocrine cells (Cheng and Leblond, 1974; Ponder et al., 1985). Generally, the differentiated enterocytes make up the majority of the cells of gut mucosa and then undergo a process of apoptosis and extrusion into the gut lumen. The actual cytodifferentiation of the gut epithelium in mice occurs at 17–18 days of gestation (Klein and McKenzie, 1983a, 1983b). In this study, we analyzed the expression profile of eIF3a during mouse development and in human colon cancer cell lines under differentiated and undifferentiated conditions. Our results indicate that eIF3a may play an important role in mouse development and in colon epithelial cell differentiation and that the decreased expression of eIF3a may be a prerequisite of intestinal epithelial cell differentiation.
erase chain reaction (PCR) product was digested with BamHI and cloned into the expression vector pGEX-4T-1. Glutathione-S-transferase fusion protein was induced with IPTG and purified using a glutathione-Sepharose 4B column (Pharmacia, Piscataway, NJ) as previously described (Zhang et al., 1996). The spectrin domain was released from the fusion protein by digestion using thrombin. Adult female rabbits were immunized subcutaneously with 500 mg of the recombinant protein representing the spectrin domain of eIF3a in complete Freund’s adjuvant. Subsequent challenges were made at approximately 4-week intervals with 500 mg each of the recombinant protein in incomplete Freund’s adjuvant. Two weeks after the final boosting, tester bleeding was conducted and 2 months after weekly bleeding, the rabbit was sacrificed for serum. All animal experiments were carried out using protocol approved by the Indiana University School of Medicine Institutional Animal Care and Research Advisory Committee. Animal mating and tissue preparation Timed mating was carried out by placing one fertile male (C57BL/ 6J) with one or two females of the same strain in a cage, and the male was removed when females were checked for the presence of vaginal plugs. Gestation day 1 was defined by the presence of a plug. Mice were housed at 72–741F and 60% humidity with 12-hr light–dark lighting cycle. Water and food were available ad libitum. Pregnant mice were killed on days 8, 10, 12, 15, and 18 of gestation by decapitation and fetuses were delivered by cesarean section. At these stages of development, fetal small intestine, stomach, lung, kidney, liver, and heart were readily discernible when viewed under the dissecting stereomicroscope (Stereomaster Zoom Microscope, Fisher Scientific). Postnatal mice were also sacrificed on days 1, 2, 3, 10, 21, and 90 by decapitation. Intestine, stomach, lung, kidney, liver, and heart were removed from fetal and postnatal mice and rinsed twice with iced phosphate-buffered saline (PBS). All samples (fetal and postnatal) were immediately frozen in liquid N2 and stored at 701C before further study. Adult mice were also sacrificed by decapitation. Intestinal tissues were harvested in cold PBS. The intestinal segments were longitudinally opened and washed twice with cold PBS to remove the contents. The mucosa was separated from the underlying muscularis by sharp dissection (Klingensmith et al., 1996; Hallonquist et al., 1998). The mucosal tissues and bases of the crypt were immediately frozen and stored in liquid N2 for further processing.
Materials and methods Immunocytochemistry staining of eIF3a in mouse intestine Materials Antibodies against actin and a-tubulin and reagents for the alkaline phosphatase (AP) and sucrase assays were purchased from Sigma (St. Louis, MO). The antibody against GAPDH was from Abcam Inc. (Cambridge, MA). The enhanced chemiluminenscence (ECL) system for Western blot analysis was from Amersham Biosciences (Piscataway, NJ). Sequi-Blot polyvinylidene difluoride (PVDF) membrane and concentrated protein assay dye reagents were from Bio-Rad (Hercules, CA). Cell culture media and reagents were from Invitrogen (Carlsbad, CA). Small interference RNA (siRNA) for eIF3a and the nonspecific control siRNAs (cat # D-001206-12-20) were synthesized by Dharmacon (Lafayette, CO). All other reagents were of molecular biology grade and obtained from Sigma or Fisher Scientific (Chicago, IL).
The adult mouse intestine was dissected in cold PBS and immediately fixed in 4% paraformaldehyde (PFA) for 3 hr at 41C. The tissue sample was next incubated in 30% sucrose at 41C overnight, embedded in OCT compound, and cut at 10 mm on a Leica cryostat (Leica Microsystems, Wetzlar, Germany). After the sections were air dried, they were blocked with 1% goat serum in PBS at room temperature for 30 min and incubated with either an affinity-purified polyclonal antibody AbD against eIF3a (1:100 dilution) or control immunoglobulin G (IgG) antibody at 41C overnight. Next, the sections were incubated with a Cy2-labeled secondary antibody for 2 hr and with nuclear dye Hoechst for 10 min at room temperature. The immuno-staining were examined under a Leica DM500 fluorescent microscope. Engineering of reporter constructs
Generation of polyclonal antibody AbS against the spectrin domain of eIF3a The cDNA encoding the spectrin domain of eIF3a was amplified using Pfu polymerase with primers 5 0 -CGGGATCCATGG CTAAACAGGTTG and 5 0 -CCAATCGATTATGCC. The polym-
The short promoter of human sucrase ( 210 to 154) was amplified from human genomic DNA using primers PhSIF 5 0 -GGCTCG AGGCTTTGAGAAATCAAAGAGTATCTGAC-3 0 and PhSIR 5 0 -GGAAGCTTAGCCTGTTCTCTTTGCTATGTTGT-3 0 with a XbaI site in PhSIF and a HindIII site in PhSIR. The PCR product
654 was cloned into pGEM T-Easy vector (Promega, Madison, WI) followed by sequencing confirmation, released by digestion with XbaI and HindIII, and subsequently cloned into pGL3 basic vector (Promega) to obtain the reporter construct, pGL-hSIP.
Caco-2 cell lysates. The reaction was detected by horseradish peroxidase-conjugated goat anti-rabbit antibody and visualized using ECL.
AP and sucrase assays Culture of HT-29 cells and sodium butyrate treatment The human colon cancer cell line HT-29 was maintained in modified Eagle’s medium supplemented with 15% fetal calf serum at 371C under humidified atmosphere containing 5% CO2. HT29 cells were seeded in 100-mm dishes at a density of 1–1.5 106 cells per dish and were maintained for 36 hr (for 48 and 72 hr treatment group), 48 hr (for 12 and 24 hr treatment group), or 72 hr (for 8 hr treatment group) before the media were replaced with fresh ones containing 7 mM sodium butyrate and cultured continuously till harvest of cells for preparation of cell lysates and determination of eIF3a and AP expressions.
Cells were harvested by trypsin digestion and washed in PBS. Cell lysate was prepared by lysing cells in Tris-mannitol buffer (2 mM Tris and 50 mM mannitol, pH value 7.1) with sonication and the lysates were cleared by centrifugation at 6,000 g for 5 min at 41C. Protein concentrations of lysates were determined using the method of Bradford (1976). AP activity was determined using an AP assay kit from Sigma. Sucrase activity was determined according to the method of Messer and Dahlqvist (1966). The enzyme activities were expressed as unit per milligram protein (U/mg) with the unit defined as the enzymatic activity that hydrolyzes 1 mmol substrate/min at 371C. The data were analyzed using Student’s t-test for statistical significance.
Culture of Caco-2 cells and transfection The human colon cancer cell line Caco-2 was maintained in modified Eagle’s medium supplemented with 10% fetal calf serum at 371C under humidified atmosphere containing 5% CO2. For confluence-induced differentiation studies, 1 106 Caco-2 cells/dish were seeded in 60 mm dishes and maintained for various times before collection for further studies. For transient transfection of pCbA-eIF3a, 1 106 cells/dish were seeded in 60 mm culture dishes and maintained for 24 hr before transfection with 2 mg pCbA vector or 4 mg pCbA-eIF3a. For luciferase reporter assay, the cells were co-transfected with 2 mg pCbA, 0.5 mg pCMV-Sports-b-gal and 1.5 mg pGL-hSIP or pGL3 basic vector control, or 4 mg pCbAeIF3a, 0.5 mg pCMV-Sports-b-gal, and 1.5 mg pGL-hSIP or pGL3 basic vector control. Cells were harvested 96 hr posttransfection and lysates were prepared for determination of expression of eIF3a and brush-boarder enzymes or luciferase activity assay as described below. siRNA of eIF3a, synthesized by Dharmacon Inc., has a sequence of 5 0 -AAGCAGCCUGCUCUGGAUGdTdT-3 0 . For transient siRNA transfection, 3 105 Caco-2 cells/well were seeded in six-well cell culture plates and were allowed to grow for 24 hr. The cells were then transfected with 0.3 mmol of eIF3a or control siRNAs using LipofectAMINEt 2000 (Invitrogen, Grand Island, NY) according to manufacturer’s recommendations. Cells were harvested 48 or 96 hr after transfection for analysis of eIF3a expression and activity and expression of brush-border enzymes. For luciferase reporter assay, the cells were transfected with pCMV-Sports-b-gal together with pGL-hSIP reporter plasmid or the control pGL3 basic vector 24 hr after siRNA transfection. Cells were incubated for 24 hr and then harvested for luciferase assay.
Luciferase reporter assay Cells transfected with reporter constructs were harvested and lysed in the Reporter Lysis Buffer (Promega). The firefly luciferase activity was determined using the Mono-Luciferase Reporter Assay Kit (Promega). The activity of b-galactosidase was measured as described previously (Dong et al., 2005) and was used as internal control to normalize transfection efficiency.
RNA purification and real-time PCR analysis Total cellular RNAs were extracted using an RNeasy Mini Kit (Qiagen, Valencia, CA). The levels of both sucrase and AP mRNAs were determined using real-time quantitative RT-PCR as described previously (Dong et al., 2005). Briefly, 1 mg of total RNAs were reverse transcribed using an iScript cDNA Synthesis Kit (Bio-Rad) and the real-time PCR were carried out in an ABI Prism@7000 Sequence Detection System (Applied Biosystems, Foster City, CA) using SYBR Green diction according to the manufacturer’s instructions. The primers used for sucrase were 5 0 -GAGGACACT GGCTTGGAGAC-3 0 (forward) and 5 0 -ATCCAGCGGGTAC AGAGATG-3 0 (reverse) and those for AP were 5 0 -GTGGATC AGGACACCTGAAGAAG-3 0 (forward) and 5 0 -GGGAATCC CCGATCCAGTAG (reverse). The threshold cycle (Ct) was defined as the PCR cycle number at which the reporter fluorescence crosses the threshold reflecting a statistically significant point above the calculated baseline. The Ct of each target product was determined and normalized against that of internal control, GAPDH. The relative mRNA level was 2DCt .
Sample preparation and Western blot analysis The frozen mouse tissues were thawed and homogenized in TNNSDS buffer (50 mM Tris-HCl, pH value 7.5, 150 mM NaCl, 0.5% Nonidet P-40, 50 mM NaF, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 0.1% sodium dodecyl sulfate [SDS], and 2 mM phenylmethylsulfonyl fluoride) and the lysates were clarified by centrifugation (10,000 g for 30 min at 41C). Protein concentrations of the lysates were determined using the method of Bradford (1976). Western blot analyses were performed as described previously (Pincheira et al., 2001b). Briefly, protein samples were separated by 8% SDS-polyacryilamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane followed by a 2-hr incubation in blocking solution (Tris-buffered saline containing 5% nonfat dried milk and 0.1% Tween 20) and a 2-hr incubation with polyclonal antibody AbS (1:5,000 dilution) for mouse tissue lysates or the affinity-purified polyclonal antibody AbD (1:750 dilution) for
Results Preparation of polyclonal antibodies against eIF3a AbD, an affinity-purified polyclonal antibody against eIF3a, has been used successfully in the past to determine the level of eIF3a protein in human tissues and cell lines (Pincheira et al., 2001a, 2001b; Dong and Zhang, 2003; Dong et al., 2004). To better serve for the current study using mouse, we generated another polyclonal antibody using the spectrin domain of eIF3a (Pincheira et al., 2001b), which is well conserved between mouse and human as an immunogen. This antibody, named
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Fig. 1 Characterization of polyclonal antibody AbS. (A) Western blot analysis of NIH3T3 cell lysates. Different amounts of cell lysates from NIH3T3 cells were separated by sodium dodecyl sulfatepolyacryilamide gel electrophoresis followed by Western blot analysis probed with AbS. GAPDH was used as loading controls. (B) Immunoprecipitation and Western blot analysis of eIF3a with AbS. Lysates from human cancer cell line H1299 were subjected to immunoprecipitation using affinity-purified AbD (lane 4) or control IgG (lane 3) followed by Western blot probed with AbS. Lanes 1 and 2 are cell lysate input probed with AbD (lane 1) or AbS (lane 2).
AbS reacts specifically with eIF3a as determined by Western blotting (Fig. 1A) and by first immunoprecipitating eIF3a with AbD followed by Western blot analysis with AbS (Fig. 1B).
EIF3a expression during mouse development We first assessed the expression profile of eIF3a in mouse fetus during development using the polyclonal antibody AbS. As shown in Figure 2A, it appears that the level of eIF3a expression in the whole fetus is high and it changed very little in the whole fetus during the development with a slight increase on the 12th day of gestation. Previously, we found that the expression of eIF3a in the adult human lung is not detectable and it is increased in human lung cancers (Pincheira et al., 2001b). It is, thus, of interest to determine the eIF3a expression profile in the developing lung of mice. As shown in Figure 2E, eIF3a is detected in the fetal lung on the 15th day of gestation and it disappeared on the 18th day before birth. On the first day after birth, eIF3a expression in the lung is elevated and it disappeared on the following days. The observation that eIF3a expression is detected in mouse fetal lung but not in the lung of late postnatal stage is consistent with our previous observation of eIF3a expression patterns in human fetal and adult lungs (Pincheira et al., 2001b). The reason for the elevated expression of eIF3a on the first day after birth is not known. However, it may be related to the stress generated from exposure of the lung to outside air after birth. We next examined the eIF3a expression profile in other mouse developing tissues. As shown in Figures
Fig. 2 Expression of eukaryotic initiation factor 3a (eIF3a) in mouse fetus and fetal tissues. Homogenates from whole mouse fetus (A), fetal and postnatal liver (B), kidney (C), heart (D), lung (E), stomach (F), intestine (G), and from intestinal mucosa and base of crypt of adult mice (H) were isolated and 10 mg of lysate in each lane was separated by sodium dodecyl sulfate-polyacryilamide gel electrophoresis followed by Western blot analysis probed with AbS. GAPDH, tubulin, or actin were used as loading controls.
2B–2D, eIF3a expression was noted throughout the fetal and postnatal stages in the developing mouse liver, kidney, and heart with a gradual decrease in expression level on the 10th (kidney) or 21st (liver and heart) day of the postnatal stage. EIF3a expression in kidney was not detectable while it can still be detected in both liver and heart on the 90th day. Interestingly, in the developing
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Fig. 3 Eukaryotic initiation factor 3a (eIF3a) immuno-staining in sections of adult mouse small intestine. Sections of frozen adult mouse intestine were incubated with eIF3a-specific antibody (A–D) or negative control immunoglobulin G (E–H) followed by staining with secondary antibody-conjugated with Cy2. Nuclei were counterstained with Hoechst. The sections were viewed under a fluores-
cent microscope for staining of Cy2 (A, D, E, and H), Hoechst (B and F), or combined (C and G). (D) and (H) are enlarged sections from (A) and (E), respectively. The asterisks indicate the reference points between (A) and (D) and between (E) and (H). Arrows in (D) indicates the eIF3a-expressing cells in the basal section of mouse intestine.
stomach (Fig. 2F) and intestine (Fig. 2G), eIF3a expression was detected only in the fetal but not in the postnatal tissues, similar to the findings of lung (see Fig. 2E). In the fetal intestine, the expression of eIF3a peaked on the 12th day and disappeared on the 18th day before birth (Fig. 2G). Similarly, the expression of eIF3a in stomach was also mainly in the fetus and it also disappeared on the 18th day before birth (Fig. 2F). Because the actual cytodifferentiation of the gut epithelium in mouse occurs on the 17th–18th day of gestation (Klein and McKenzie, 1983a, 1983b), the decreased eIF3a expression on the 18th day suggests that the eIF3a expression may inversely correlate with cell differentiation in the gut. To test this possibility, we isolated basal and mucosal cells from adult mouse intestine and determined the expression level of eIF3a. It is known that cells in the mucosa of the small intestine usually possess a high degree of differentiation whereas the base of the crypt mainly consists of poorly differentiated cells. As shown in Figure 2H, the eIF3a expression is clearly detected in the undifferentiated basal but not in the differentiated mucosal epithelial cells. To confirm this observation, we performed an immunocytochemistry staining of cross sections of mouse small intestine. As shown in Figure 3, eIF3a appears to be expressed only in the undifferentiated basal region of intestine (Cy2 staining in the basal region). Thus, likely eIF3a expression negatively correlates with differentiation of epithelial cells in intestinal tract.
EIF3a expression in relationship with colon epithelial cell differentiation To further examine the relationship between eIF3a expression and cell differentiation, we took advantage of the human colon cancer cell line HT-29 which, upon treatment with sodium butyrate, differentiates in culture and expresses brush-border enzymes such as AP (Boren et al., 2003). For this purpose, we first treated HT-29 cells with 7 mM sodium butyrate for different times and the cells were then collected for analysis of the expression of AP and eIF3a. As shown in Figure 4A, AP expression was increased at 48 hr following butyrate treatment, suggesting that HT-29 cells began to differentiate at this time. However, eIF3a expression began to decrease at 12 hr following butyrate treatment (Fig. 4B), about 36 hr before cell differentiation. These findings suggest that the decreased eIF3a expression occurs before cell differentiation and, thus, it may play a role in HT-29 cell differentiation. To further test the role of eIF3a in colon epithelial cell differentiation and to rule out the effect of chemical agents such as sodium butyrate on eIF3a expression, we used another human colon cancer cell line Caco-2, which also has much better transfection efficiency than HT-29 cells. For in vitro studies of differentiation, Caco-2 represents one of the best models. It spontaneously differentiates into enterocytes with a small bowel-like phenotype, as indicated by dome formation, presence of microvilli, and expression of brush-border enzymes
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Fig. 4 Expression of eukaryotic initiation factor 3a (eIF3a) and alkaline phosphatase (AP) in differentiating HT-29 cells. HT-29 cells were cultured in 100-mm dishes at a density of 1–1.5 106 cells per dish followed by treatment with sodium butyrate for various times. Cell lysates were then prepared for analysis of AP activity (A) and expression level of eIF3a (B).
such as AP and sucrase after confluence (Pinto et al., 1983; Zweibaum and Chantret, 1989; Markowitz et al., 1995; Ding et al., 1998). We first examined the correlation between eIF3a expression and Caco-2 cell differentiation by determining AP and sucrase activity. For this purpose, 1 106 Caco2 cells/dish were seeded in 60-mm dishes and cultured till the cells reached near and beyond confluence. The cells were collected for analysis of eIF3a expression and indicators of differentiation, AP and sucrase. As shown in Figure 5A, marked increases in the activity of AP and sucrase were observed on day 3 post-confluence when the cell number reached plateau (Fig. 5B) and it continued to increase up to 12 days postconfluence, suggesting that the postconfluent Caco-2 cells were differentiated. The expression of eIF3a, in contrast, experienced a dramatic decrease following confluence at day 0 (Fig. 5C) before the increase in AP and sucrase activities. These findings, consistent with that of HT-29 cells, suggest that the decreased eIF3a expression occurs before Caco-2 cell differentiation and it may play a role in enterocyte differentiation. We next tested if suppressing eIF3a expression would induce differentiation of Caco-2 cells without the cells reaching confluence. For this purpose, we used eIF3a siRNAs to knock down eIF3a expression in Caco-2 cells cultured at low density (see Materials and methods for design). As shown in Figure 6A, transfection of the eIF3a siRNA into Caco-2 cells drastically reduced the eIF3a expression as compared with the control nonspecific siRNA. Interestingly, the decreased eIF3a expression by siRNA significantly increased activity (Fig. 6B) and the mRNA level (Fig. 6C) of both AP and sucrase without the cells reaching confluence. Thus, it is possible that the reduction in eIF3a expression level alone may have induced Caco-2 cell differentiation.
Fig. 5 Expression of eukaryotic initiation factor 3a (eIF3a) and brush-border enzymes in differentiating Caco-2 cells. Caco-2 cells were seeded at 1 106 cells/dish in 60-mm culture dishes and maintained for various times. Cell lysates were then prepared for analysis of alkaline phosphatase (AP) and sucrase (Suc) activities (A) and the expression level of eIF3a (C) using Western blot probed with AbD. (B) Shows the growth curve of Caco-2 cells seeded at 1 106 cells/60 mm dish. Cell number was counted using a hemocytometer. GAPDH was used as a loading control for Western blot.
To further test this possibility, we examined if enforced overexpression of eIF3a would block the confluence-induced Caco-2 cell differentiation. We transiently transfected pCbA-eIF3a into Caco-2 cells as previously described (Dong and Zhang, 2003) to overexpress ectopic eIF3a and then allowed cells to reach confluence before determining the AP and sucrase activities. As shown in Figure 7A, the level of endogenous eIF3a in the vector-transfected cells decreased at 96 hr following transfection suggesting that the cells have reached confluence. However, the eIF3a level in the pCbA-eIF3a-transfected cells at 96 hr following transfection remained high due to the enforced expression of the ectopic eIF3a. We next determined the activities and mRNA levels of AP and sucrase in these cells and found that the enforced expression of ectopic eIF3a significantly suppressed the increase in activities and mRNA level of both enzymes induced by confluence (Figs. 7B–7E). Taken together, these data suggest that eIF3a may play an important role in Caco-2 cell differentiation. In order to explore the possible mechanism that eIF3a influences the expression of the differentiation marker enzymes, we engineered luciferase reporter con-
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Fig. 6 Expression of eukaryotic initiation factor 3a (eIF3a) and brush-border enzymes in Caco-2 cells treated with small interference RNA (siRNA) of eIF3a. For siRNA treatment, 3 105 Caco2 cells/well were seeded in six-well culture plates and grown for 24 hr followed by transfection with siRNA of eIF3a or nonspecific control siRNA (Scr). Cells were then harvested 48 hr posttransfec-
tion. Cell lysates were prepared for analysis of eIF3a expression (A) and alkaline phosphatase (AP) and sucrase activities (B). The data shown were from average of six independent experiments (for AP, p 0.001; for sucrase, p 0.004). Total RNAs were also prepared for analysis of AP and sucrase mRNA levels using real-time PCR (C).
struct by cloning the short promoter ( 210 to 154) of human sucrase gene in pGL3 basic vector. It has been reported previously that this short promoter contains HIF-1a and GATA sites (Krasinski et al., 2001; Escaffit et al., 2005). This construct was transfected into preconfluent Caco-2 cells transfected with eIF3a siRNA or cotransfected with pCbA-eIF3a into the confluent Caco-2 cells as described above to determine the effect of eIF3a expression on the promoter activity. As shown in Figure 8, decreasing eIF3a expression by siRNA (Fig. 8A) or up-regulating eIF3a expression by ectopic cDNA (Fig. 8B) had no effect on the activity of the short promoter of sucrase. These results suggest that the effect of eIF3a on the expression of sucrase may not be through the HIF-1a and GATA transcription factors.
In this study, we for the first time investigated the early temporal expression profile of the eIF3a gene in the
developing mouse small intestine, stomach, liver, kidney, and heart and the role of eIF3a in regulating cell differentiation. We found that eIF3a is expressed mainly in the fetal tissues and its expression negatively regulates cell differentiation. There are at least two distinct expression patterns of eIF3a during mouse development. While it is clear that the eIF3a expression ceased in fetal lung, intestine, and stomach on the 18th day before birth, the expression of eIF3a persisted until the 21st and 90th day in the postnatal liver, kidney, and heart; however, the level of eIF3a decreased on the 10th and 21st day in these tissues. Previously, using dot blot analysis we found that the eIF3a expression at the mRNA level is high in most human fetal tissues including heart, kidney, liver, and lung (Pincheira et al., 2001b); however, its expression in the corresponding adult tissues is much lower. The results from the current study of mouse tissues are consistent with these findings. These observations suggest that the potential effect of eIF3a expression on development may be different in different organs.
Fig. 7 Expression of eukaryotic initiation factor 3a (eIF3a) and brush-border enzymes in Caco-2 cells with ectopic overexpression of eIF3a. For ectopic overexpression, 1 106 Caco-2 cells/well were seeded in 60 mm culture dishes and cultured for 24 hr followed by transfection with pCbA-eIF3a or vector control. Cells were harvested 96 hr posttransfection and lysates were prepared for analysis
of eIF3a expression (A) or activities of alkaline phosphatase (AP) (B) and sucrase (C) as described in Materials and methods. The data shown were averages of six independent experiments (p 0.001). Total RNAs were also prepared for the determination of mRNA levels of AP (D) and sucrase (E) using real-time quantitative RT-PCR.
Discussion
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Fig. 8 Effect of eukaryotic initiation factor 3a (eIF3a) on the short promoter activity of human sucrase gene. Reporter construct, pGLhSIP, was transfected into preconfluent Caco-2 cells that were transfected with eIF3a small interference RNA (siRNA) (A) or cotransfected with pCbA-eIF3a into the confluent Caco-2 cells (B).
The cells were then harvested and lysed for luciferase assay. The luciferase activities from pGL-hSIP (black bar) were normalized to the cotransfected b-galactosidase for transfection efficiency and then to that of cells transfected with pGL3 basic vector control (open bar).
The early cessation of eIF3a expression in the intestinal tract roughly corresponds to initiation of intestinal morphogenesis and actual cytodifferentiation of the intestinal epithelium, suggesting that intestinal cell differentiation is associated with eIF3a expression (see discussion below). It is noteworthy that eIF3a expression in mouse lung experienced an elevation on the first day after birth. Similarly, a slight increase was also observed in stomach on the first day after birth. The reason for these acute changes in eIF3a expression is currently unknown. However, it may be due to the stress generated from the sudden exposure of the lung to outside air and stomach to milk in the newborn animals. Together, these studies suggest that the expression of eIF3a may play important roles in regulating cell differentiation and development of mammals. It should be noted, however, that in the adult intestinal tissue eIF3a is expressed only in the undifferentiated cells. Because the undifferentiated cell composition of these tissues were so low that eIF3a was undetectable in the complete tissue lysates by Western blot analysis. However, removing the mucosal tissue helped enrich the basal undifferentiated cells for detection of eIF3a by Western blot. This observation also suggests that the lack of eIF3a detection in total lysate of other postnatal tissues of mouse cannot rule out the possibility that these tissues also contain small number of undifferentiated cells that express eIF3a. Previously, elevated eIF3a expression has been observed in several types of cancers including lung (Pincheira et al., 2001b), breast (Bachmann et al., 1997), esophagus (Chen and Burger, 1999), cervix (Dellas et al., 1998), and stomach (Chen and Burger, 2004). Bachmann et al. (1997) also found that eIF3a expression decreased in several cell types upon induction of differentiation. These observations suggest that the
eIF3a expression is positively related to carcinogenesis and negatively related to differentiation. In a previous study, we have shown that eIF3a may function as a proto-oncogene and the suppression of eIF3a expression could reverse the malignant phenotype of lung and breast cancer cells (Dong et al., 2004). In this study, we showed that eIF3a is also likely involved in mediating HT-29 and Caco-2 cell differentiation. The expression of eIF3a drastically decreased in HT-29 cells at 12 hr following sodium butyrate treatment and in Caco-2 cells on day 0 in the postconfluence period. However, the expression of differentiation indicator enzymes increased only at 48 hr following butyrate treatment for HT-29 cells and on day 3 postconfluence for Caco-2 cells, suggesting that the decrease in eIF3a expression likely plays an important role in mediating the condition-induced differentiation. Indeed, enforced expression of an ectopic eIF3a in Caco-2 cells prevented confluence-induced differentiation and knocking down the expression of eIF3a using siRNA alone may have caused Caco-2 cell differentiation without the cells reaching confluence. AP and sucrase have been used as markers of Caco-2 cell differentiation (Ding et al., 1998). Because eIF3a may be involved in regulating the translation of a subset of genes (Dong and Zhang, 2003, 2006; Dong et al., 2004), the increased activity level of AP and sucrase may be due to their altered translation by eIF3a and, thus, it may not reflect the differentiation level of Caco2 cells. Although we cannot rule out this possibility, we believe that this possibility is unlikely. Firstly, the translation of many mRNAs are down-regulated and very few genes are up-regulated with the decreased expression of eIF3a (Dong and Zhang, 2003; Dong et al., 2004). Secondly, the drastic post-confluence increase of the AP and sucrase in Caco-2 cells is much delayed
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compared with the drastic decrease in eIF3a level (Fig. 5), which is inconsistent with the direct involvement of eIF3a in translational regulation of AP and sucrase expressions. This is also supported by our observation that the drastic increase in AP expression in HT-29 cells following butyrate treatment was also much delayed compared with the drastic decrease in eIF3a level (Fig. 4). Finally, it has been shown previously that the increase in AP and sucrase activity in differentiated cells is due to their increased accumulation of mRNA (Beaulieu and Quaroni, 1991; Halline et al., 1994; Wang et al., 2001). Consistently, we found that the mRNA level of AP and sucrase appeared to change by changing eIF3a expression, likely due to changes in cell differentiation. The mechanism of eIF3a involvement in development and differentiation is not known. However, the finding that eIF3a may function as a regulator controlling the translation of a subset of mRNAs (Dong and Zhang, 2003, 2006; Dong et al., 2004) suggests that the expression of important protein factors for development and differentiation may be under eIF3a control. It has been thought that both ubiquitous and tissue-specific transcription factors are important (Simon and Gordon, 1995; Traber and Silberg, 1996). Perhaps, the expression of these transcription factors is under eIF3a control. The finding that the transcription of AP and sucrase was up-regulated with the decreased eIF3a expression and vice versa is consistent with this possibility despite the fact that the short promoter of sucrase was not affected by alteration of eIF3a expression. It has also been found that terminal differentiation is associated with cell cycle arrest (Decker, 1995; Andres and Walsh, 1996; Bocchia et al., 1997; Morse et al., 1997). We found previously that eIF3a mediates mimosineinduced G1 arrest and its expression is decreased in G1 phase (Dong and Zhang, 2003). Thus, it is also tempting to propose that the decreased eIF3a expression causes G1 arrest, which then leads to terminal differentiation. We are currently investigating these possibilities. Acknowledgments This work was supported in part by National Institutes of Health grants CA94961 and CA120221 (J. T. Z.), EY017061 (X. Z.), and by the Department of Defense grants DAMD17-02-1-0073 and DAMD17-03-1-0566 (J. T. Z.). Z. D. and Y. Y. were supported, in part, by the NRSA T32 DK07519 and T32HL07910 from the National Institutes of Health, respectively.
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Pincheira, R., Chen, Q. and Zhang, J.T. (2001b) Identification of a 170-kda protein over-expressed in lung cancers. Br J Cancer 84:1520–1527. Pinto, M., Robine-Leon, S., Appay, M.D., Kedinger, M., Triadou, N., Dussaulx, E., Lacroix, B., Simon-Assmann, P., Haffen, K., Fogh, J. and Zweibaum, A. (1983) Enterocyte-like differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture. Biol Cell 47:323–330. Ponder, B.A., Schmidt, G.H., Wilkinson, M.M., Wood, M.J., Monk, M. and Reid, A. (1985) Derivation of mouse intestinal crypts from single progenitor cells. Nature 313:689–691. Simon, T.C. and Gordon, J.I. (1995) Intestinal epithelial cell differentiation: new insights from mice, flies and nematodes. Curr Opin Genet Dev 5:577–586. Traber, P.G. and Silberg, D.G. (1996) Intestine-specific gene transcription. Annu Rev Physiol 58:275–297. Wang, Q., Wang, X., Hernandez, A., Kim, S. and Evers, B.M. (2001) Inhibition of the phosphatidylinositol 3-kinase pathway contributes to HT29 and Caco-2 intestinal cell differentiation. Gastroenterology 120:1381–1392. Zhang, M., Wang, G., Shapiro, A. and Zhang, J.T. (1996) Topological folding and proteolysis profile of P-glycoprotein in membranes of multidrug-resistant cells: implications for the drug-transport mechanism. Biochemistry 35:9728–9736. Zweibaum, A. and Chantret, I. (1989) Human colon carcinoma cell lines as in vitro models for the study of intestinal cell differentiation. In: Smith, M.W. and Sepulveda, F.V. eds. Adaptation and development of gastrointestinal function. Manchester University Press, Manchester, UK, pp. 103–112.
OR IGI N A L A R T IC L E
Zhaoqian Liu . Zizheng Dong . Zuocheng Yang . Qun Chen . Yi Pan . Youyun Yang . Ping Cui . Xin Zhang . Jian-Ting Zhang
Role of eIF3a (eIF3 p170) in intestinal cell differentiation and its association with early development
Received July 21, 2006; accepted in revised form December 21, 2006
Abstract Eukaryotic initiation factor 3a (eIF3a) has been suggested to play a regulatory role in mRNA translation. Decreased eIF3a expression has been observed in differentiated cells while higher levels have been observed in cancer cells. However, whether eIF3a plays any role in differentiation and development is currently unknown. Here, we investigated eIF3a expression during mouse development and its role in differentiation of colon epithelial cells. We found that eIF3a expression was higher in fetal tissues compared with postnatal ones. Its expression in intestine, stomach, and
lung abruptly stopped on the 18th day in gestation but persisted in liver, kidney, and heart throughout the postnatal stage at decreased levels. Similarly, eIF3a expression in colon cancer cell lines, HT-29 and Caco-2, drastically decreased prior to differentiation. Enforced eIF3a expression inhibited while knocking it down using small interference RNA promoted Caco-2 differentiation. Thus, eIF3a may play some roles in development and differentiation and that the decreased eIF3a expression may be a pre-requisite of intestinal epithelial cell differentiation.
Zhaoqian Liu 1,2 Zizheng Dong1 Qun Chen Youyun . ) Yang Ping Cui Jian-Ting Zhang (* Department of Pharmacology and Toxicology Walther Oncology Center Walther Cancer Institute Indiana University School of Medicine 1044 W. Walnut Street Indianapolis, IN 46202, U.S.A. Tel: 11 317 278 4503 Fax: 11 317 274 8046 E-mail: [email protected]
Key words translational regulation protein synthesis initiation factor cancer Caco-2
Zuocheng Yang1 Department of Pediatrics The Third Xiangya Hospital Central South University Changsha, Hunan 410013, China Yi Pan Xin Zhang Department of Medical and Molecular Genetics Indiana University School of Medicine 975 W. Walnut Street Indianapolis, IN 46202, U.S.A. 1
These authors contributed equally.
2 Present address: Institute of Clinical Pharmacology, Xiangya Medical School, Central South University, Changsha, Hunan 410078, China.
Introduction The initiation step in eukaryotic protein synthesis is generally promoted by eukaryotic initiation factors (eIFs) (Dong and Zhang, 2006). Among these factors, eIF3 is the largest complex consisting of 13 subunits and it plays an essential role in translation by binding directly to the 40S ribosomal subunit and promoting formation of the 40S pre-initiation complex (Dong and Zhang, 2006). However, one of the 13 subunits of eIF3, eIF3a (p170), has been thought not to be required for the function of eIF3 in translation initiation (Chaudhuri et al., 1997) and it may play some role as a regulator for translation of a subset of mRNAs (Dong and Zhang, 2003, 2006; Dong et al., 2004). It has been found that the expression of eIF3a is elevated in human cancers of lung (Pincheira et al., 2001b), breast (Bachmann et al., 1997), esophagus (Chen and Burger, 1999), cervix (Dellas et al., 1998), and stomach (Chen and Burger, 2004). It has also been found that the expression of eIF3a is decreased in differentiated cells in culture (Bachmann
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et al., 1997). While we have shown that eIF3a expression plays some roles in cell proliferation and carcinogenesis and suppressing eIF3a expression could reverse the malignant phenotype of lung and breast cancer cells (Dong et al., 2004), it remains unknown whether eIF3a plays any role in cell differentiation and development. The gastrointestinal tract represents one of the best models for studying mechanisms of development and differentiation. In mouse, the intestinal tube forms at approximately 8–10 days of gestation (Kedinger, 1994). The digestive organs including liver, stomach, duodenum, and pancreas originate from the embryonic gut. The mammalian intestinal mucosa generally undergoes a process of continued renewal characterized by active proliferation of stem cells localized near the base of the crypts, progression of these cells up the crypt-villas with cessation of proliferation, and subsequent differentiation into one of the four primary cell types including goblet, enterocytes, paneth, and enteroendocrine cells (Cheng and Leblond, 1974; Ponder et al., 1985). Generally, the differentiated enterocytes make up the majority of the cells of gut mucosa and then undergo a process of apoptosis and extrusion into the gut lumen. The actual cytodifferentiation of the gut epithelium in mice occurs at 17–18 days of gestation (Klein and McKenzie, 1983a, 1983b). In this study, we analyzed the expression profile of eIF3a during mouse development and in human colon cancer cell lines under differentiated and undifferentiated conditions. Our results indicate that eIF3a may play an important role in mouse development and in colon epithelial cell differentiation and that the decreased expression of eIF3a may be a prerequisite of intestinal epithelial cell differentiation.
erase chain reaction (PCR) product was digested with BamHI and cloned into the expression vector pGEX-4T-1. Glutathione-S-transferase fusion protein was induced with IPTG and purified using a glutathione-Sepharose 4B column (Pharmacia, Piscataway, NJ) as previously described (Zhang et al., 1996). The spectrin domain was released from the fusion protein by digestion using thrombin. Adult female rabbits were immunized subcutaneously with 500 mg of the recombinant protein representing the spectrin domain of eIF3a in complete Freund’s adjuvant. Subsequent challenges were made at approximately 4-week intervals with 500 mg each of the recombinant protein in incomplete Freund’s adjuvant. Two weeks after the final boosting, tester bleeding was conducted and 2 months after weekly bleeding, the rabbit was sacrificed for serum. All animal experiments were carried out using protocol approved by the Indiana University School of Medicine Institutional Animal Care and Research Advisory Committee. Animal mating and tissue preparation Timed mating was carried out by placing one fertile male (C57BL/ 6J) with one or two females of the same strain in a cage, and the male was removed when females were checked for the presence of vaginal plugs. Gestation day 1 was defined by the presence of a plug. Mice were housed at 72–741F and 60% humidity with 12-hr light–dark lighting cycle. Water and food were available ad libitum. Pregnant mice were killed on days 8, 10, 12, 15, and 18 of gestation by decapitation and fetuses were delivered by cesarean section. At these stages of development, fetal small intestine, stomach, lung, kidney, liver, and heart were readily discernible when viewed under the dissecting stereomicroscope (Stereomaster Zoom Microscope, Fisher Scientific). Postnatal mice were also sacrificed on days 1, 2, 3, 10, 21, and 90 by decapitation. Intestine, stomach, lung, kidney, liver, and heart were removed from fetal and postnatal mice and rinsed twice with iced phosphate-buffered saline (PBS). All samples (fetal and postnatal) were immediately frozen in liquid N2 and stored at 701C before further study. Adult mice were also sacrificed by decapitation. Intestinal tissues were harvested in cold PBS. The intestinal segments were longitudinally opened and washed twice with cold PBS to remove the contents. The mucosa was separated from the underlying muscularis by sharp dissection (Klingensmith et al., 1996; Hallonquist et al., 1998). The mucosal tissues and bases of the crypt were immediately frozen and stored in liquid N2 for further processing.
Materials and methods Immunocytochemistry staining of eIF3a in mouse intestine Materials Antibodies against actin and a-tubulin and reagents for the alkaline phosphatase (AP) and sucrase assays were purchased from Sigma (St. Louis, MO). The antibody against GAPDH was from Abcam Inc. (Cambridge, MA). The enhanced chemiluminenscence (ECL) system for Western blot analysis was from Amersham Biosciences (Piscataway, NJ). Sequi-Blot polyvinylidene difluoride (PVDF) membrane and concentrated protein assay dye reagents were from Bio-Rad (Hercules, CA). Cell culture media and reagents were from Invitrogen (Carlsbad, CA). Small interference RNA (siRNA) for eIF3a and the nonspecific control siRNAs (cat # D-001206-12-20) were synthesized by Dharmacon (Lafayette, CO). All other reagents were of molecular biology grade and obtained from Sigma or Fisher Scientific (Chicago, IL).
The adult mouse intestine was dissected in cold PBS and immediately fixed in 4% paraformaldehyde (PFA) for 3 hr at 41C. The tissue sample was next incubated in 30% sucrose at 41C overnight, embedded in OCT compound, and cut at 10 mm on a Leica cryostat (Leica Microsystems, Wetzlar, Germany). After the sections were air dried, they were blocked with 1% goat serum in PBS at room temperature for 30 min and incubated with either an affinity-purified polyclonal antibody AbD against eIF3a (1:100 dilution) or control immunoglobulin G (IgG) antibody at 41C overnight. Next, the sections were incubated with a Cy2-labeled secondary antibody for 2 hr and with nuclear dye Hoechst for 10 min at room temperature. The immuno-staining were examined under a Leica DM500 fluorescent microscope. Engineering of reporter constructs
Generation of polyclonal antibody AbS against the spectrin domain of eIF3a The cDNA encoding the spectrin domain of eIF3a was amplified using Pfu polymerase with primers 5 0 -CGGGATCCATGG CTAAACAGGTTG and 5 0 -CCAATCGATTATGCC. The polym-
The short promoter of human sucrase ( 210 to 154) was amplified from human genomic DNA using primers PhSIF 5 0 -GGCTCG AGGCTTTGAGAAATCAAAGAGTATCTGAC-3 0 and PhSIR 5 0 -GGAAGCTTAGCCTGTTCTCTTTGCTATGTTGT-3 0 with a XbaI site in PhSIF and a HindIII site in PhSIR. The PCR product
654 was cloned into pGEM T-Easy vector (Promega, Madison, WI) followed by sequencing confirmation, released by digestion with XbaI and HindIII, and subsequently cloned into pGL3 basic vector (Promega) to obtain the reporter construct, pGL-hSIP.
Caco-2 cell lysates. The reaction was detected by horseradish peroxidase-conjugated goat anti-rabbit antibody and visualized using ECL.
AP and sucrase assays Culture of HT-29 cells and sodium butyrate treatment The human colon cancer cell line HT-29 was maintained in modified Eagle’s medium supplemented with 15% fetal calf serum at 371C under humidified atmosphere containing 5% CO2. HT29 cells were seeded in 100-mm dishes at a density of 1–1.5 106 cells per dish and were maintained for 36 hr (for 48 and 72 hr treatment group), 48 hr (for 12 and 24 hr treatment group), or 72 hr (for 8 hr treatment group) before the media were replaced with fresh ones containing 7 mM sodium butyrate and cultured continuously till harvest of cells for preparation of cell lysates and determination of eIF3a and AP expressions.
Cells were harvested by trypsin digestion and washed in PBS. Cell lysate was prepared by lysing cells in Tris-mannitol buffer (2 mM Tris and 50 mM mannitol, pH value 7.1) with sonication and the lysates were cleared by centrifugation at 6,000 g for 5 min at 41C. Protein concentrations of lysates were determined using the method of Bradford (1976). AP activity was determined using an AP assay kit from Sigma. Sucrase activity was determined according to the method of Messer and Dahlqvist (1966). The enzyme activities were expressed as unit per milligram protein (U/mg) with the unit defined as the enzymatic activity that hydrolyzes 1 mmol substrate/min at 371C. The data were analyzed using Student’s t-test for statistical significance.
Culture of Caco-2 cells and transfection The human colon cancer cell line Caco-2 was maintained in modified Eagle’s medium supplemented with 10% fetal calf serum at 371C under humidified atmosphere containing 5% CO2. For confluence-induced differentiation studies, 1 106 Caco-2 cells/dish were seeded in 60 mm dishes and maintained for various times before collection for further studies. For transient transfection of pCbA-eIF3a, 1 106 cells/dish were seeded in 60 mm culture dishes and maintained for 24 hr before transfection with 2 mg pCbA vector or 4 mg pCbA-eIF3a. For luciferase reporter assay, the cells were co-transfected with 2 mg pCbA, 0.5 mg pCMV-Sports-b-gal and 1.5 mg pGL-hSIP or pGL3 basic vector control, or 4 mg pCbAeIF3a, 0.5 mg pCMV-Sports-b-gal, and 1.5 mg pGL-hSIP or pGL3 basic vector control. Cells were harvested 96 hr posttransfection and lysates were prepared for determination of expression of eIF3a and brush-boarder enzymes or luciferase activity assay as described below. siRNA of eIF3a, synthesized by Dharmacon Inc., has a sequence of 5 0 -AAGCAGCCUGCUCUGGAUGdTdT-3 0 . For transient siRNA transfection, 3 105 Caco-2 cells/well were seeded in six-well cell culture plates and were allowed to grow for 24 hr. The cells were then transfected with 0.3 mmol of eIF3a or control siRNAs using LipofectAMINEt 2000 (Invitrogen, Grand Island, NY) according to manufacturer’s recommendations. Cells were harvested 48 or 96 hr after transfection for analysis of eIF3a expression and activity and expression of brush-border enzymes. For luciferase reporter assay, the cells were transfected with pCMV-Sports-b-gal together with pGL-hSIP reporter plasmid or the control pGL3 basic vector 24 hr after siRNA transfection. Cells were incubated for 24 hr and then harvested for luciferase assay.
Luciferase reporter assay Cells transfected with reporter constructs were harvested and lysed in the Reporter Lysis Buffer (Promega). The firefly luciferase activity was determined using the Mono-Luciferase Reporter Assay Kit (Promega). The activity of b-galactosidase was measured as described previously (Dong et al., 2005) and was used as internal control to normalize transfection efficiency.
RNA purification and real-time PCR analysis Total cellular RNAs were extracted using an RNeasy Mini Kit (Qiagen, Valencia, CA). The levels of both sucrase and AP mRNAs were determined using real-time quantitative RT-PCR as described previously (Dong et al., 2005). Briefly, 1 mg of total RNAs were reverse transcribed using an iScript cDNA Synthesis Kit (Bio-Rad) and the real-time PCR were carried out in an ABI Prism@7000 Sequence Detection System (Applied Biosystems, Foster City, CA) using SYBR Green diction according to the manufacturer’s instructions. The primers used for sucrase were 5 0 -GAGGACACT GGCTTGGAGAC-3 0 (forward) and 5 0 -ATCCAGCGGGTAC AGAGATG-3 0 (reverse) and those for AP were 5 0 -GTGGATC AGGACACCTGAAGAAG-3 0 (forward) and 5 0 -GGGAATCC CCGATCCAGTAG (reverse). The threshold cycle (Ct) was defined as the PCR cycle number at which the reporter fluorescence crosses the threshold reflecting a statistically significant point above the calculated baseline. The Ct of each target product was determined and normalized against that of internal control, GAPDH. The relative mRNA level was 2DCt .
Sample preparation and Western blot analysis The frozen mouse tissues were thawed and homogenized in TNNSDS buffer (50 mM Tris-HCl, pH value 7.5, 150 mM NaCl, 0.5% Nonidet P-40, 50 mM NaF, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 0.1% sodium dodecyl sulfate [SDS], and 2 mM phenylmethylsulfonyl fluoride) and the lysates were clarified by centrifugation (10,000 g for 30 min at 41C). Protein concentrations of the lysates were determined using the method of Bradford (1976). Western blot analyses were performed as described previously (Pincheira et al., 2001b). Briefly, protein samples were separated by 8% SDS-polyacryilamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane followed by a 2-hr incubation in blocking solution (Tris-buffered saline containing 5% nonfat dried milk and 0.1% Tween 20) and a 2-hr incubation with polyclonal antibody AbS (1:5,000 dilution) for mouse tissue lysates or the affinity-purified polyclonal antibody AbD (1:750 dilution) for
Results Preparation of polyclonal antibodies against eIF3a AbD, an affinity-purified polyclonal antibody against eIF3a, has been used successfully in the past to determine the level of eIF3a protein in human tissues and cell lines (Pincheira et al., 2001a, 2001b; Dong and Zhang, 2003; Dong et al., 2004). To better serve for the current study using mouse, we generated another polyclonal antibody using the spectrin domain of eIF3a (Pincheira et al., 2001b), which is well conserved between mouse and human as an immunogen. This antibody, named
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Fig. 1 Characterization of polyclonal antibody AbS. (A) Western blot analysis of NIH3T3 cell lysates. Different amounts of cell lysates from NIH3T3 cells were separated by sodium dodecyl sulfatepolyacryilamide gel electrophoresis followed by Western blot analysis probed with AbS. GAPDH was used as loading controls. (B) Immunoprecipitation and Western blot analysis of eIF3a with AbS. Lysates from human cancer cell line H1299 were subjected to immunoprecipitation using affinity-purified AbD (lane 4) or control IgG (lane 3) followed by Western blot probed with AbS. Lanes 1 and 2 are cell lysate input probed with AbD (lane 1) or AbS (lane 2).
AbS reacts specifically with eIF3a as determined by Western blotting (Fig. 1A) and by first immunoprecipitating eIF3a with AbD followed by Western blot analysis with AbS (Fig. 1B).
EIF3a expression during mouse development We first assessed the expression profile of eIF3a in mouse fetus during development using the polyclonal antibody AbS. As shown in Figure 2A, it appears that the level of eIF3a expression in the whole fetus is high and it changed very little in the whole fetus during the development with a slight increase on the 12th day of gestation. Previously, we found that the expression of eIF3a in the adult human lung is not detectable and it is increased in human lung cancers (Pincheira et al., 2001b). It is, thus, of interest to determine the eIF3a expression profile in the developing lung of mice. As shown in Figure 2E, eIF3a is detected in the fetal lung on the 15th day of gestation and it disappeared on the 18th day before birth. On the first day after birth, eIF3a expression in the lung is elevated and it disappeared on the following days. The observation that eIF3a expression is detected in mouse fetal lung but not in the lung of late postnatal stage is consistent with our previous observation of eIF3a expression patterns in human fetal and adult lungs (Pincheira et al., 2001b). The reason for the elevated expression of eIF3a on the first day after birth is not known. However, it may be related to the stress generated from exposure of the lung to outside air after birth. We next examined the eIF3a expression profile in other mouse developing tissues. As shown in Figures
Fig. 2 Expression of eukaryotic initiation factor 3a (eIF3a) in mouse fetus and fetal tissues. Homogenates from whole mouse fetus (A), fetal and postnatal liver (B), kidney (C), heart (D), lung (E), stomach (F), intestine (G), and from intestinal mucosa and base of crypt of adult mice (H) were isolated and 10 mg of lysate in each lane was separated by sodium dodecyl sulfate-polyacryilamide gel electrophoresis followed by Western blot analysis probed with AbS. GAPDH, tubulin, or actin were used as loading controls.
2B–2D, eIF3a expression was noted throughout the fetal and postnatal stages in the developing mouse liver, kidney, and heart with a gradual decrease in expression level on the 10th (kidney) or 21st (liver and heart) day of the postnatal stage. EIF3a expression in kidney was not detectable while it can still be detected in both liver and heart on the 90th day. Interestingly, in the developing
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Fig. 3 Eukaryotic initiation factor 3a (eIF3a) immuno-staining in sections of adult mouse small intestine. Sections of frozen adult mouse intestine were incubated with eIF3a-specific antibody (A–D) or negative control immunoglobulin G (E–H) followed by staining with secondary antibody-conjugated with Cy2. Nuclei were counterstained with Hoechst. The sections were viewed under a fluores-
cent microscope for staining of Cy2 (A, D, E, and H), Hoechst (B and F), or combined (C and G). (D) and (H) are enlarged sections from (A) and (E), respectively. The asterisks indicate the reference points between (A) and (D) and between (E) and (H). Arrows in (D) indicates the eIF3a-expressing cells in the basal section of mouse intestine.
stomach (Fig. 2F) and intestine (Fig. 2G), eIF3a expression was detected only in the fetal but not in the postnatal tissues, similar to the findings of lung (see Fig. 2E). In the fetal intestine, the expression of eIF3a peaked on the 12th day and disappeared on the 18th day before birth (Fig. 2G). Similarly, the expression of eIF3a in stomach was also mainly in the fetus and it also disappeared on the 18th day before birth (Fig. 2F). Because the actual cytodifferentiation of the gut epithelium in mouse occurs on the 17th–18th day of gestation (Klein and McKenzie, 1983a, 1983b), the decreased eIF3a expression on the 18th day suggests that the eIF3a expression may inversely correlate with cell differentiation in the gut. To test this possibility, we isolated basal and mucosal cells from adult mouse intestine and determined the expression level of eIF3a. It is known that cells in the mucosa of the small intestine usually possess a high degree of differentiation whereas the base of the crypt mainly consists of poorly differentiated cells. As shown in Figure 2H, the eIF3a expression is clearly detected in the undifferentiated basal but not in the differentiated mucosal epithelial cells. To confirm this observation, we performed an immunocytochemistry staining of cross sections of mouse small intestine. As shown in Figure 3, eIF3a appears to be expressed only in the undifferentiated basal region of intestine (Cy2 staining in the basal region). Thus, likely eIF3a expression negatively correlates with differentiation of epithelial cells in intestinal tract.
EIF3a expression in relationship with colon epithelial cell differentiation To further examine the relationship between eIF3a expression and cell differentiation, we took advantage of the human colon cancer cell line HT-29 which, upon treatment with sodium butyrate, differentiates in culture and expresses brush-border enzymes such as AP (Boren et al., 2003). For this purpose, we first treated HT-29 cells with 7 mM sodium butyrate for different times and the cells were then collected for analysis of the expression of AP and eIF3a. As shown in Figure 4A, AP expression was increased at 48 hr following butyrate treatment, suggesting that HT-29 cells began to differentiate at this time. However, eIF3a expression began to decrease at 12 hr following butyrate treatment (Fig. 4B), about 36 hr before cell differentiation. These findings suggest that the decreased eIF3a expression occurs before cell differentiation and, thus, it may play a role in HT-29 cell differentiation. To further test the role of eIF3a in colon epithelial cell differentiation and to rule out the effect of chemical agents such as sodium butyrate on eIF3a expression, we used another human colon cancer cell line Caco-2, which also has much better transfection efficiency than HT-29 cells. For in vitro studies of differentiation, Caco-2 represents one of the best models. It spontaneously differentiates into enterocytes with a small bowel-like phenotype, as indicated by dome formation, presence of microvilli, and expression of brush-border enzymes
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Fig. 4 Expression of eukaryotic initiation factor 3a (eIF3a) and alkaline phosphatase (AP) in differentiating HT-29 cells. HT-29 cells were cultured in 100-mm dishes at a density of 1–1.5 106 cells per dish followed by treatment with sodium butyrate for various times. Cell lysates were then prepared for analysis of AP activity (A) and expression level of eIF3a (B).
such as AP and sucrase after confluence (Pinto et al., 1983; Zweibaum and Chantret, 1989; Markowitz et al., 1995; Ding et al., 1998). We first examined the correlation between eIF3a expression and Caco-2 cell differentiation by determining AP and sucrase activity. For this purpose, 1 106 Caco2 cells/dish were seeded in 60-mm dishes and cultured till the cells reached near and beyond confluence. The cells were collected for analysis of eIF3a expression and indicators of differentiation, AP and sucrase. As shown in Figure 5A, marked increases in the activity of AP and sucrase were observed on day 3 post-confluence when the cell number reached plateau (Fig. 5B) and it continued to increase up to 12 days postconfluence, suggesting that the postconfluent Caco-2 cells were differentiated. The expression of eIF3a, in contrast, experienced a dramatic decrease following confluence at day 0 (Fig. 5C) before the increase in AP and sucrase activities. These findings, consistent with that of HT-29 cells, suggest that the decreased eIF3a expression occurs before Caco-2 cell differentiation and it may play a role in enterocyte differentiation. We next tested if suppressing eIF3a expression would induce differentiation of Caco-2 cells without the cells reaching confluence. For this purpose, we used eIF3a siRNAs to knock down eIF3a expression in Caco-2 cells cultured at low density (see Materials and methods for design). As shown in Figure 6A, transfection of the eIF3a siRNA into Caco-2 cells drastically reduced the eIF3a expression as compared with the control nonspecific siRNA. Interestingly, the decreased eIF3a expression by siRNA significantly increased activity (Fig. 6B) and the mRNA level (Fig. 6C) of both AP and sucrase without the cells reaching confluence. Thus, it is possible that the reduction in eIF3a expression level alone may have induced Caco-2 cell differentiation.
Fig. 5 Expression of eukaryotic initiation factor 3a (eIF3a) and brush-border enzymes in differentiating Caco-2 cells. Caco-2 cells were seeded at 1 106 cells/dish in 60-mm culture dishes and maintained for various times. Cell lysates were then prepared for analysis of alkaline phosphatase (AP) and sucrase (Suc) activities (A) and the expression level of eIF3a (C) using Western blot probed with AbD. (B) Shows the growth curve of Caco-2 cells seeded at 1 106 cells/60 mm dish. Cell number was counted using a hemocytometer. GAPDH was used as a loading control for Western blot.
To further test this possibility, we examined if enforced overexpression of eIF3a would block the confluence-induced Caco-2 cell differentiation. We transiently transfected pCbA-eIF3a into Caco-2 cells as previously described (Dong and Zhang, 2003) to overexpress ectopic eIF3a and then allowed cells to reach confluence before determining the AP and sucrase activities. As shown in Figure 7A, the level of endogenous eIF3a in the vector-transfected cells decreased at 96 hr following transfection suggesting that the cells have reached confluence. However, the eIF3a level in the pCbA-eIF3a-transfected cells at 96 hr following transfection remained high due to the enforced expression of the ectopic eIF3a. We next determined the activities and mRNA levels of AP and sucrase in these cells and found that the enforced expression of ectopic eIF3a significantly suppressed the increase in activities and mRNA level of both enzymes induced by confluence (Figs. 7B–7E). Taken together, these data suggest that eIF3a may play an important role in Caco-2 cell differentiation. In order to explore the possible mechanism that eIF3a influences the expression of the differentiation marker enzymes, we engineered luciferase reporter con-
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Fig. 6 Expression of eukaryotic initiation factor 3a (eIF3a) and brush-border enzymes in Caco-2 cells treated with small interference RNA (siRNA) of eIF3a. For siRNA treatment, 3 105 Caco2 cells/well were seeded in six-well culture plates and grown for 24 hr followed by transfection with siRNA of eIF3a or nonspecific control siRNA (Scr). Cells were then harvested 48 hr posttransfec-
tion. Cell lysates were prepared for analysis of eIF3a expression (A) and alkaline phosphatase (AP) and sucrase activities (B). The data shown were from average of six independent experiments (for AP, p 0.001; for sucrase, p 0.004). Total RNAs were also prepared for analysis of AP and sucrase mRNA levels using real-time PCR (C).
struct by cloning the short promoter ( 210 to 154) of human sucrase gene in pGL3 basic vector. It has been reported previously that this short promoter contains HIF-1a and GATA sites (Krasinski et al., 2001; Escaffit et al., 2005). This construct was transfected into preconfluent Caco-2 cells transfected with eIF3a siRNA or cotransfected with pCbA-eIF3a into the confluent Caco-2 cells as described above to determine the effect of eIF3a expression on the promoter activity. As shown in Figure 8, decreasing eIF3a expression by siRNA (Fig. 8A) or up-regulating eIF3a expression by ectopic cDNA (Fig. 8B) had no effect on the activity of the short promoter of sucrase. These results suggest that the effect of eIF3a on the expression of sucrase may not be through the HIF-1a and GATA transcription factors.
In this study, we for the first time investigated the early temporal expression profile of the eIF3a gene in the
developing mouse small intestine, stomach, liver, kidney, and heart and the role of eIF3a in regulating cell differentiation. We found that eIF3a is expressed mainly in the fetal tissues and its expression negatively regulates cell differentiation. There are at least two distinct expression patterns of eIF3a during mouse development. While it is clear that the eIF3a expression ceased in fetal lung, intestine, and stomach on the 18th day before birth, the expression of eIF3a persisted until the 21st and 90th day in the postnatal liver, kidney, and heart; however, the level of eIF3a decreased on the 10th and 21st day in these tissues. Previously, using dot blot analysis we found that the eIF3a expression at the mRNA level is high in most human fetal tissues including heart, kidney, liver, and lung (Pincheira et al., 2001b); however, its expression in the corresponding adult tissues is much lower. The results from the current study of mouse tissues are consistent with these findings. These observations suggest that the potential effect of eIF3a expression on development may be different in different organs.
Fig. 7 Expression of eukaryotic initiation factor 3a (eIF3a) and brush-border enzymes in Caco-2 cells with ectopic overexpression of eIF3a. For ectopic overexpression, 1 106 Caco-2 cells/well were seeded in 60 mm culture dishes and cultured for 24 hr followed by transfection with pCbA-eIF3a or vector control. Cells were harvested 96 hr posttransfection and lysates were prepared for analysis
of eIF3a expression (A) or activities of alkaline phosphatase (AP) (B) and sucrase (C) as described in Materials and methods. The data shown were averages of six independent experiments (p 0.001). Total RNAs were also prepared for the determination of mRNA levels of AP (D) and sucrase (E) using real-time quantitative RT-PCR.
Discussion
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Fig. 8 Effect of eukaryotic initiation factor 3a (eIF3a) on the short promoter activity of human sucrase gene. Reporter construct, pGLhSIP, was transfected into preconfluent Caco-2 cells that were transfected with eIF3a small interference RNA (siRNA) (A) or cotransfected with pCbA-eIF3a into the confluent Caco-2 cells (B).
The cells were then harvested and lysed for luciferase assay. The luciferase activities from pGL-hSIP (black bar) were normalized to the cotransfected b-galactosidase for transfection efficiency and then to that of cells transfected with pGL3 basic vector control (open bar).
The early cessation of eIF3a expression in the intestinal tract roughly corresponds to initiation of intestinal morphogenesis and actual cytodifferentiation of the intestinal epithelium, suggesting that intestinal cell differentiation is associated with eIF3a expression (see discussion below). It is noteworthy that eIF3a expression in mouse lung experienced an elevation on the first day after birth. Similarly, a slight increase was also observed in stomach on the first day after birth. The reason for these acute changes in eIF3a expression is currently unknown. However, it may be due to the stress generated from the sudden exposure of the lung to outside air and stomach to milk in the newborn animals. Together, these studies suggest that the expression of eIF3a may play important roles in regulating cell differentiation and development of mammals. It should be noted, however, that in the adult intestinal tissue eIF3a is expressed only in the undifferentiated cells. Because the undifferentiated cell composition of these tissues were so low that eIF3a was undetectable in the complete tissue lysates by Western blot analysis. However, removing the mucosal tissue helped enrich the basal undifferentiated cells for detection of eIF3a by Western blot. This observation also suggests that the lack of eIF3a detection in total lysate of other postnatal tissues of mouse cannot rule out the possibility that these tissues also contain small number of undifferentiated cells that express eIF3a. Previously, elevated eIF3a expression has been observed in several types of cancers including lung (Pincheira et al., 2001b), breast (Bachmann et al., 1997), esophagus (Chen and Burger, 1999), cervix (Dellas et al., 1998), and stomach (Chen and Burger, 2004). Bachmann et al. (1997) also found that eIF3a expression decreased in several cell types upon induction of differentiation. These observations suggest that the
eIF3a expression is positively related to carcinogenesis and negatively related to differentiation. In a previous study, we have shown that eIF3a may function as a proto-oncogene and the suppression of eIF3a expression could reverse the malignant phenotype of lung and breast cancer cells (Dong et al., 2004). In this study, we showed that eIF3a is also likely involved in mediating HT-29 and Caco-2 cell differentiation. The expression of eIF3a drastically decreased in HT-29 cells at 12 hr following sodium butyrate treatment and in Caco-2 cells on day 0 in the postconfluence period. However, the expression of differentiation indicator enzymes increased only at 48 hr following butyrate treatment for HT-29 cells and on day 3 postconfluence for Caco-2 cells, suggesting that the decrease in eIF3a expression likely plays an important role in mediating the condition-induced differentiation. Indeed, enforced expression of an ectopic eIF3a in Caco-2 cells prevented confluence-induced differentiation and knocking down the expression of eIF3a using siRNA alone may have caused Caco-2 cell differentiation without the cells reaching confluence. AP and sucrase have been used as markers of Caco-2 cell differentiation (Ding et al., 1998). Because eIF3a may be involved in regulating the translation of a subset of genes (Dong and Zhang, 2003, 2006; Dong et al., 2004), the increased activity level of AP and sucrase may be due to their altered translation by eIF3a and, thus, it may not reflect the differentiation level of Caco2 cells. Although we cannot rule out this possibility, we believe that this possibility is unlikely. Firstly, the translation of many mRNAs are down-regulated and very few genes are up-regulated with the decreased expression of eIF3a (Dong and Zhang, 2003; Dong et al., 2004). Secondly, the drastic post-confluence increase of the AP and sucrase in Caco-2 cells is much delayed
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compared with the drastic decrease in eIF3a level (Fig. 5), which is inconsistent with the direct involvement of eIF3a in translational regulation of AP and sucrase expressions. This is also supported by our observation that the drastic increase in AP expression in HT-29 cells following butyrate treatment was also much delayed compared with the drastic decrease in eIF3a level (Fig. 4). Finally, it has been shown previously that the increase in AP and sucrase activity in differentiated cells is due to their increased accumulation of mRNA (Beaulieu and Quaroni, 1991; Halline et al., 1994; Wang et al., 2001). Consistently, we found that the mRNA level of AP and sucrase appeared to change by changing eIF3a expression, likely due to changes in cell differentiation. The mechanism of eIF3a involvement in development and differentiation is not known. However, the finding that eIF3a may function as a regulator controlling the translation of a subset of mRNAs (Dong and Zhang, 2003, 2006; Dong et al., 2004) suggests that the expression of important protein factors for development and differentiation may be under eIF3a control. It has been thought that both ubiquitous and tissue-specific transcription factors are important (Simon and Gordon, 1995; Traber and Silberg, 1996). Perhaps, the expression of these transcription factors is under eIF3a control. The finding that the transcription of AP and sucrase was up-regulated with the decreased eIF3a expression and vice versa is consistent with this possibility despite the fact that the short promoter of sucrase was not affected by alteration of eIF3a expression. It has also been found that terminal differentiation is associated with cell cycle arrest (Decker, 1995; Andres and Walsh, 1996; Bocchia et al., 1997; Morse et al., 1997). We found previously that eIF3a mediates mimosineinduced G1 arrest and its expression is decreased in G1 phase (Dong and Zhang, 2003). Thus, it is also tempting to propose that the decreased eIF3a expression causes G1 arrest, which then leads to terminal differentiation. We are currently investigating these possibilities. Acknowledgments This work was supported in part by National Institutes of Health grants CA94961 and CA120221 (J. T. Z.), EY017061 (X. Z.), and by the Department of Defense grants DAMD17-02-1-0073 and DAMD17-03-1-0566 (J. T. Z.). Z. D. and Y. Y. were supported, in part, by the NRSA T32 DK07519 and T32HL07910 from the National Institutes of Health, respectively.
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