Characterization and regulation of E2F activity during Caco-2 cell differentiation

Qingming Ding, Qingding Wang, Zizheng Dong, and B. Mark Evers

Department of Surgery, The University of Texas Medical Branch, Galveston, Texas 77555


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The specific mechanisms controlling intestinal cell differentiation remain largely undefined. The retinoblastoma (Rb) proteins (pRb, p130, and p107) appear crucial to the terminal differentiation process of certain cells through their association and repression of E2F transcription factors. We have examined the expression of pRb-related proteins p130 and p107 as well as the regulation of E2F during spontaneous differentiation of the Caco-2 intestinal cell line. Nuclear protein levels of p130 and p107 were increased with Caco-2 differentiation. Induction of a slower-migrating E2F complex was noted in postconfluent (i.e., differentiated) Caco-2 cells; p130 protein was the predominant component of this E2F complex with a minor contribution from cyclin-dependent kinase-2. A small component of p107 binding was identified by deoxycholate release gel shift assays. In contrast, no pRb binding to E2F was noted in Caco-2 cells. In addition to increased association with p130, E2F-4 phosphorylation was markedly decreased in differentiated Caco-2 cells, whereas E2F protein levels remained unchanged. Taken together, our findings suggest that the regulation of E2F function may be an important contributing factor in the cell cycle block and spontaneous differentiation of Caco-2 cells. This regulation of E2F occurs most likely through its increased association with p130 as well as decreased phosphorylation.

gut differentiation; cell cycle; retinoblastoma-related proteins


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE EPITHELIUM of the gastrointestinal tract is composed of numerous cell types and is in a constant state of self-renewal (32). This process involves a highly regimented and orderly progression of proliferation and subsequent differentiation as cells ascend the crypt-villus axis (17, 44). The stem cell, localized to the crypt, differentiates into one of four primary cell types, with absorptive enterocytes making up the majority of terminally differentiated cells in the villus (5, 33, 35). This process of terminal differentiation is associated with an irreversible cell cycle withdrawal and the expression of tissue-specific genes, such as the brush-border enzymes sucrase-isomaltase (SI) and alkaline phosphatase (44). Within 3-5 days, the enterocytes reach the villus tip and are extruded into the lumen (21, 34). The cellular mechanisms regulating this orderly series of events, which are critical for normal intestinal homeostasis and development, remain largely undefined.

The terminal differentiation process in a number of cell types is regulated by the sequential activation and inactivation of proteins that control the mammalian cell cycle at two key checkpoints, the G1/S and the G2/M transition points (29, 30). Progression through the cell cycle is controlled by the activation of a highly conserved family of cyclin-dependent kinases (Cdks), which bind their regulatory subunits (i.e., the cyclins) (24, 28). The activities of these Cdks can be inhibited by the binding of Cdk inhibitory proteins, which results in a cell cycle block and differentiation in certain cell types (11, 40). One family of Cdk inhibitors includes p21Waf1/Cip1, p27Kip1/Pic2, and p57Kip2, which are universal inhibitors of the cyclin-Cdk complexes (11, 24, 40). Using the human colon cancer cell line Caco-2, which spontaneously differentiates to a small bowel-like phenotype, as noted by dome formation, the presence of microvilli, and the expression of brush-border enzymes after confluency is reached (31, 45, 51), we have shown an early induction of p21Waf1/Cip1 occurring in Caco-2 cells on postconfluent day 3 that precedes induction of SI gene expression (12); this early induction of p21Waf1/Cip1 in Caco-2 cells has been further confirmed by Gartel et al. (15) and Abraham et al. (1). Moreover, we have shown that Caco-2 cells undergo a relative G1/S block and cessation of proliferation at 3 days postconfluency associated with suppression of Cdk2 and Cdk4 activities (8). Our findings, however, indicate that p21Waf1/Cip1 may only be partially responsible for Cdk2 suppression at postconfluent day 3, suggesting that other mechanisms contribute to this cell cycle block and subsequent differentiation.

Another class of negative cell cycle regulators includes the retinoblastoma (Rb) protein (pRb) and the pRb-related proteins p130 and p107, which can regulate cell cycle progression through their association with and repression of the E2F family of transcription factors (6, 25, 42, 47). Members of the E2F family function, at least in part, as heterodimers composed of an E2F and a DP subunit (2, 26, 27, 38). Six members of the E2F family, designated E2F-1-E2F-6, and their heterodimeric partners, DP-1, -2, and -3, have been identified (6, 16, 42). Complexes containing E2F-1, -2, or -3 associated with pRb, but not p107 or p130, in vivo (10, 22). In contrast, complexes containing E2F-4 or -5 reportedly bind preferentially to p107 and p130 (3, 18, 46). The interaction of p130 and p107 with the E2F proteins has been implicated in cellular proliferation and differentiation. For example, p130-E2F binding appears to be most prominent in quiescent and various growth-arrested or differentiated cell types, such as muscle, neurons, and peripheral T cells, suggesting that the formation of the p130-E2F complex is a necessary event for the terminal differentiation process in these cells (7, 19, 23, 36, 37, 41, 43). In addition to the association with the pRb proteins, other mechanisms that can regulate E2F function during differentiation include changes in E2F protein levels and differential phosphorylation of the E2F protein (9, 41, 49).

Expression of p107 has been detected in early fetal gut but is only present at background levels in the late fetal stage (20). Chandrasekaran et al. (4) showed that an increase of hypophosphorylated pRb in the nucleus may be associated with the differentiated state. However, the specific role of pRb, the pRb-related proteins (p130 and p107), and the E2F family of proteins in intestinal cell differentiation has not been entirely defined. The purpose of our present study was to characterize the expression of pRb, p130, and p107 proteins during Caco-2 cell differentiation. In addition, we examined the regulation and function of E2F family members at the level of protein-protein interactions, protein expression, and phosphorylation. We demonstrate that Caco-2 cell differentiation is associated with an increase of p130 and p107 protein levels in the nucleus. In addition, E2F DNA binding complexes were regulated during Caco-2 cell differentiation, with the p130 protein noted to be the major component of the E2F complex associated with differentiation. The levels of E2F proteins remained unchanged; however, E2F-4 phosphorylation markedly decreased with Caco-2 cell differentiation. Therefore, our present study suggests that interaction of E2F with p130 and decreased E2F phosphorylation may contribute to the terminal differentiation process in intestinal cells.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Radioactive compounds were purchased from DuPont-New England Nuclear (Boston, MA). Immobilon-P nylon membranes for Western blots were purchased from Millipore (Bedford, MA), and X-ray film was purchased from Eastman Kodak (Rochester, NY). An oligonucleotide containing two E2F consensus binding sites (5'-AGCTTGTTT<UNL>TCGCGC</UNL>TTAAATTTGAGAAAG<UNL>GGCGCGAA</UNL>ACTAGTCA-3') was synthesized by Oligos Etc. (Wilsonville, OR). All the antibodies used in this study were from Santa Cruz Biotechnologies (Santa Cruz, CA) and include antibodies to pRb (sc-1538), p130 (sc-317), p107 (sc-318), E2F-2 (sc-633), E2F-4 (sc-1082), DP-1 (sc-610), DP-2 (sc-829), Cdk2 (sc-163), and beta -actin (sc-1616). The enhanced chemiluminescence (ECL) system for Western immunoblot analysis was from Amersham (Arlington Heights, IL). The concentrated protein assay dye reagent was purchased from Bio-Rad Laboratories (Hercules, CA). Protein A-Sepharose (CL-4B) was from Pharmacia Biotech. Tissue culture media and reagents were obtained from GIBCO-BRL (Grand Island, NY). All other reagents were of molecular biology grade and were purchased from Sigma Chemical (St. Louis, MO) or Amresco (Solon, OH).

Cell culture. The human colon cancer cell line Caco-2, obtained from American Type Culture Collection (Rockville, MD), was maintained in MEM supplemented with 15% (vol/vol) FCS. All cells are maintained in a humidified atmosphere of 95% air-5% CO2 at 37°C. Studies were performed on cells at various times before they reached confluency (preconfluent) or at various time points after confluency.

Western blotting. Western blotting was performed as described previously (8, 12). Briefly, cytoplasmic and nuclear protein (40 µg) was denatured by heating to 95°C for 10 min in SDS sample buffer [50 mM Tris (pH 6.8), 100 mM dithiothreitol (DTT), 2% SDS, 0.1% bromphenol blue, and 10% glycerol] before fractionation on an 8% SDS-polyacrylamide gel and then electroblotted to Immobilon-P nylon membranes. Filters were incubated overnight at 4°C in blocking solution (Tris-buffered saline containing 5% nonfat dry milk and 0.1% Tween 20) and then for 3 h with the primary antibody. Filters were incubated with a horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG as a secondary antibody for 1 h. After four final washes, the immune complexes were visualized using ECL detection.

Preparation of nuclear extracts and electrophoretic mobility shift assays. The cytoplasmic and nuclear extracts were prepared from pre- and postconfluent Caco-2 cells according to the procedure described by Schreiber et al. (39). Electrophoretic mobility shift assays (EMSAs) were performed as described previously (13). Binding reactions consisted of 10 µg of nuclear extracts incubated for 20 min at room temperature in the presence of 12.5 mM HEPES (pH 7.9), 100 mM KCl, 10% glycerol, 0.1 mM EDTA, 0.75 mM DTT, 0.2 mM phenylmethylsulfonyl fluoride, 3 µg of poly(dI · dC), and 0.5 ng of the 32P-labeled E2F probe. Competition binding experiments were performed by first incubating the unlabeled E2F oligonucleotide at 100-fold molar excess with the nuclear extracts and binding buffer for 10 min on ice. The labeled probe was then added, and incubation was continued for 15 min at room temperature. The reaction mixture was loaded onto 5% nondenaturing polyacrylamide gels and resolved by electrophoresis at 200 V for 2-3 h. For antibody studies, 2 µl of antiserum were added during the preincubation for 20 min at room temperature before the addition of labeled probe. The reaction mixtures were then allowed to incubate for an additional 30 min at room temperature before electrophoresis on a 5% nondenaturing polyacrylamide gel for 4-5 h. To determine free E2F from the E2F complexes, nuclear extracts were incubated with 0.1% deoxycholate (DOC) for 20 min on ice, and then NP-40 was added at a final concentration of 1.2%. Mobility shift assay reactions were incubated at room temperature for 30 min, and then samples were subjected to electrophoresis at 200 V for 2-3 h. The gels were subsequently dried and autoradiographed at -70°C with an intensifying screen.

DOC release EMSAs. DOC release EMSAs were performed essentially as described by Shin et al. (41). Nuclear extracts (200 µg) from preconfluent and postconfluent (0, 3, and 6 days) Caco-2 cells were incubated with 5 µl of p107-, p130-, or pRb-specific antiserum for 1 h at 4°C. Protein A-Sepharose beads (10 µl) were added to each tube, and the tubes were incubated at 4°C for an additional 2 h. Beads were washed three times with HEPES-buffered solution [20 mM HEPES (pH 7.4), 40 mM KCl, 1 mM MgCl2, 0.1 EDTA] and then incubated with 10 µl of 20 mM HEPES (pH 7.4) that contained DOC (0.8%, wt/vol) for 10 min on ice. Next, 10 µl of 2.5% (wt/vol) NP-40 was added to the tubes to a final concentration of 1.25% (wt/vol) NP-40. After centrifugation, 7 µl of supernatant were used in the presence or absence of the unlabeled E2F oligonucleotide (100-fold molar excess as competitor) and analyzed by EMSA.

In vivo labeling and immunochemical methods. Phosphorylation of E2F was measured by immunoprecipitation of preconfluent and 6-day postconfluent Caco-2 cells labeled with 32Pi (1 mCi/ml) in phosphate-free medium for 2 h. Cells were washed three times and then lysed with TNN buffer [50 mM Tris · HCl (pH 7.4), 120 mM NaCl, 5 mM EDTA, 0.5% NP-40, 50 mM NaF, 0.2 mM sodium orthovanadate, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml of aprotinin]. Total lysates (4 × 106 counts/min for each sample) were immunoprecipitated with 5 µl of anti-E2F-4 or anti-p130 and 20 µl of protein A-Sepharose beads. Beads were then washed five times with TNN buffer, and bound protein was released by boiling in the presence of 50 µl of 2% SDS and 0.1 mM DTT for 5 min. After it was boiled, the supernatant was diluted to 1 ml with TNN buffer and immunoprecipitated again with anti-E2F-4 or preimmune serum. Immunoprecipitates were analyzed by SDS-PAGE. The gels were dried and autoradiographed at -70°C with an intensifying screen.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Nuclear protein levels of p130 and p107 increase with Caco-2 cell differentiation. The human colon cancer cell line Caco-2 spontaneously differentiates into an enterocyte-like phenotype after reaching confluency (31, 45, 51). Previously, we showed that the growth of Caco-2 cells reaches a plateau at 3 days postconfluency, which is associated with a G1 cell cycle arrest, an increase in the level of Cdk inhibitor p21Waf1/Cip1, and a marked decrease in Cdk activity (8, 12); Caco-2 cell differentiation occurred after 3 days postconfluency, as noted by an increase in levels of SI and alkaline phosphatase enzyme activities. Our findings indicate, however, that the induction of p21Waf1/Cip1 was only partially responsible for the Cdk2 suppression, suggesting that other factors contribute to Caco-2 cell arrest and differentiation (8).

To determine whether protein levels of p130 and p107 were increased with Caco-2 cell differentiation, nuclear and cytoplasmic extracts obtained from preconfluent, confluent, and postconfluent (3 and 6 days) Caco-2 cells were assessed by Western blot analysis (Fig. 1). An increase in p130 and p107 nuclear protein levels was apparent by day 0 (100% confluency) compared with preconfluent levels; expression levels remained elevated in the 3- and 6-day postconfluent extracts compared with preconfluent extracts. Cytoplasmic levels of the p130 and p107 proteins decreased from day 0 to day 3, suggesting that translocation from the cytoplasm to the nucleus may account for the increased nuclear levels. Levels of pRb decreased in postconfluent cytoplasmic extracts; however, this decrease was not associated with concomitant increases in nuclear pRb. The blot was stripped and reprobed with beta -actin to confirm equal loading and intact protein. Taken together with our findings of increased p130 and p107 mRNA levels, the increases of nuclear p130 and p107 further indicate a potential contributory role for these proteins in Caco-2 cell differentiation.


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Fig. 1.   Western immunoblot analysis of p130, p107, and retinoblastoma (pRb) proteins in differentiating Caco-2 cells. Cytoplasmic and nuclear extracts from preconfluent (Pre) or postconfluent (Post; 0, 3, and 6 days postconfluency) Caco-2 cells were resolved by SDS-PAGE, transferred to a nylon membrane, and probed with anti-p130, anti-p107, anti-Rb, and anti-actin. Complexes were visualized using enhanced chemiluminescence detection (Amersham).

Analysis of E2F binding during Caco-2 differentiation. The function of the E2F transcription factor appears to be regulated during the cell cycle through interactions with pRb-related proteins and with certain cyclins and Cdks (6, 25, 27, 38, 42, 47). Changes in the steady-state protein levels of p130 and p107 suggested that E2F activity may be regulated by binding to the pRb-related proteins during Caco-2 cell differentiation.

To characterize E2F activity in differentiating Caco-2 cells, we first examined changes in E2F binding activity. Nuclear extracts from preconfluent, confluent, and postconfluent (3 and 6 days) Caco-2 cells were incubated with a synthetic oligonucleotide containing two E2F consensus binding sites (50) and analyzed by EMSA (Fig. 2A). Four protein-DNA complexes were detected in Caco-2 cell nuclear extracts (complexes A-D). As Caco-2 cells differentiate, the signal intensity of the slower-migrating complex (complex A) increased by day 0 (100% confluency), with further increases noted on days 3 and 6 postconfluency. The intensity of complex B was increased slightly. Complexes C and D remained relatively unchanged throughout the time course. The specificity of binding was confirmed by competition with a molar excess (100-fold) of unlabeled E2F. The two faster-migrating complexes (C and D) represent free E2F, whereas the two slower-migrating complexes (A and B) appear to consist of an E2F complex, which was disrupted by treating the extracts with the detergent DOC (Fig. 2B). Collectively, these findings identify regulation of E2F binding during spontaneous Caco-2 cell differentiation, as noted by the induction of a slower-migrating DNA-protein complex.



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Fig. 2.   Electrophoretic mobility shift assays (EMSAs) assessing E2F binding activity in differentiating Caco-2 cells. Nuclear extracts (10 µg) from preconfluent or postconfluent (0, 3, and 6 days) Caco-2 cells were mixed with 32P-labeled E2F oligonucleotide in absence (A) or presence (B) of deoxycholate (DOC, 0.1%) and analyzed by PAGE. Unlabeled E2F oligonucleotide (100-fold molar excess) was used as a competitor (lanes 5 and 8 in A; lane 9 in B). Four complexes were noted (A-D).

Differentiation of Caco-2 is associated with induction of p130-E2F complex. To determine the composition of this complex, which is induced with Caco-2 differentiation, EMSAs were repeated using antibodies to p130, p107, and pRb. When anti-p130 was added to the nuclear protein, a supershifted complex that included the majority of complex A was noted; the remaining three complexes were not affected (Fig. 3A). In contrast, addition of antibodies to p107 or pRb failed to alter the protein-DNA complexes (data not shown). Because Cdk2 can bind to the p130-E2F complex (48), the experiment was repeated using antisera to Cdk2; two minor supershifted complexes were detected (Fig. 3B). Therefore, these results indicate that the majority of the upper complex is composed of p130-E2F with a minor component of Cdk2 binding. Furthermore, these findings suggest that p130 is the primary pRb-related protein that regulates E2F activity during Caco-2 cell differentiation.



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Fig. 3.   Identification of p130 as major component of complex A in differentiating Caco-2 cells. Nuclear extracts (10 µg), prepared from preconfluent (Pre) and postconfluent (0, 3, and 6 days) Caco-2 cells, were preincubated with antisera to p130 (A) or Cdk2 (B) before addition of labeled E2F oligonucleotide. Supershifted complexes were noted with antisera to p130 and, to a lesser degree, Cdk2. Unlabeled E2F, added in molar excess, was used as a competitor to ensure binding specificity.

To further confirm the presence of p130 in the E2F complexes, p130 immunoprecipitates were treated with DOC and analyzed by EMSA. DOC preferentially releases proteins (e.g., E2F bound to p130) but does not significantly perturb the antibody-p130 interaction (50); the released E2F activity can then be detected by EMSA. E2F DNA binding activity in the p130 immune complexes increases in differentiating Caco-2 cells (Fig. 4A), thus further confirming the presence of p130 in association with E2F and suggesting an important role of p130 in the regulation of E2F activity during Caco-2 cell differentiation. No p107-DNA binding complex was detected by the supershift assay with differentiation, and, in agreement with these results, E2F DNA binding activity in the p107 immunoprecipitates decreased with differentiation, suggesting that p107 may only bind a small fraction of E2F in Caco-2 cells (Fig. 4B). Moreover, DOC release EMSAs with anti-pRb failed to detect E2F activity (data not shown), thus further suggesting that pRb does not interact with E2F in Caco-2 cells.


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Fig. 4.   DOC release EMSA. Nuclear extracts (200 µg) from preconfluent or postconfluent (0, 3, and 6 days) Caco-2 cells were immunoprecipitated with antisera to p130 (A) or p107 (B). Immune complexes were treated with detergent DOC (0.8%). E2F activity released from anti-p130 and anti-p107 immune complexes was analyzed by EMSA. Lane 5, unlabeled E2F oligonucleotide added as a competitor at 100-fold molar excess.

Phosphorylation of E2F-4 decreases with Caco-2 cell differentiation. In addition to regulation of E2F by protein-protein interactions, the activity of E2F can be regulated by the total amount of E2F protein and by differential phosphorylation (9, 25, 41, 47, 49). We next examined the changes in protein levels and phosphorylation of E2F with spontaneous differentiation of Caco-2. The protein levels of E2F-1, E2F-2, and E2F-4 were unchanged in nuclear and cytoplasmic extracts (Fig. 5). Although DP-1 protein decreases with differentiation, DP-2 levels in nuclear extracts were increased with Caco-2 cell differentiation. The significance of the changes in the levels of DP-1 and -2 with differentiation is not known.


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Fig. 5.   Western immunoblot analysis of E2F (E2F-1, -2, and -4) and DP (DP-1 and -2) proteins in differentiating Caco-2 cells. Cytoplasmic (100 µg) and nuclear (40 µg) extracts from preconfluent or postconfluent (0, 3, and 6 days) Caco-2 cells were resolved by SDS-PAGE, transferred to a nylon membrane, and probed with anti-E2F-1, anti-E2F-2, anti-E2F-4, anti-DP-1, and anti-DP-2 antibodies. Complexes were visualized by enhanced chemiluminescence detection.

E2F-4 is the only member of the E2F family that could be detected in the E2F binding complex (Fig. 6A); therefore, we determined the phosphorylation of E2F-4 by immunoprecipitation from 32P-labeled preconfluent and 6-day postconfluent Caco-2 cells (Fig. 6B). Preimmune IgG was used as a control; no signals were detected in the immunoprecipitation with the control IgG (Fig. 6B, lanes 1 and 2). There was a decrease in phosphorylation of E2F protein of 6-day postconfluent Caco-2 cells compared with preconfluent cells (Fig. 6B, lanes 3 and 4). Therefore, phosphorylated E2F levels were markedly decreased with Caco-2 differentiation, despite no change in E2F protein levels, suggesting a role of phosphorylation in the regulation of E2F activity during Caco-2 cell differentiation.



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Fig. 6.   Phosphorylation of E2F in differentiating Caco-2 cells. A: nuclear extracts (10 µg) from preconfluent or postconfluent (0, 3, and 6 days) Caco-2 cells were precipitated with antisera to E2F-4 before addition of labeled E2F oligonucleotide. A supershifted complex was noted with E2F-4 antisera. Unlabeled E2F, added in molar excess, was used as a competitor. B: E2F phosphorylation was measured by immunoprecipitation of extracts from preconfluent and 6-day postconfluent Caco-2 cells. Labeled Caco-2 cell lysates (4 × 106 cpm) were immunoprecipitated with anti-E2F-4 antibody and then precipitated again with preimmune serum (IgG) or anti-E2F-4. Immunoprecipitates were resolved by SDS-PAGE.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In our present study, we have analyzed the expression of the pRb proteins and the regulation of E2F function during spontaneous differentiation of the Caco-2 intestinal cell line to an enterocyte-like phenotype. Protein levels of p130 and p107 increased in the nuclear fractions with Caco-2 cell differentiation. In addition, we demonstrate regulation of E2F protein in postconfluent (i.e., differentiated) Caco-2 cells. A marked increase in a slower-migrating E2F binding complex was noted with differentiation; this binding complex was composed primarily of p130-E2F. Thus, despite an increase in p130 and p107 protein levels, p130 appears to be the predominant pRb-related protein in the regulation of E2F during the terminal differentiation process in the Caco-2 intestinal cell line.

The pRb, p130, and p107 proteins share sequence homology and can regulate cell proliferation (25, 47). Recent studies suggest that, in certain cells, there is a differential regulation of the p130 and p107 proteins with differentiation. In our present study, we demonstrate an increase in p130 nuclear protein levels in postconfluent Caco-2 cells occurring at a time point we previously showed to be associated with cell cycle block and the switch to a differentiated phenotype (8, 12). Consistent with these findings, induction of p130 has been noted with muscle cell differentiation, 3T3-L1 adipogenesis, and retinoic acid-induced differentiation of the embryonal carcinoma cell line P19 into a neuronal cell phenotype (7, 19, 23, 36). In addition, the subcellular change in p130 protein levels from the cytoplasm to the nucleus lends further support for a potential role of this protein in the regulation of Caco-2 cell differentiation.

The Rb proteins can regulate E2F activity through the differential binding to members of the E2F family; the result of this interaction is the regulation of cell cycle progression (6, 27, 38, 42). Changes in the expression levels of the pRb-related proteins suggested that E2F activity is regulated during Caco-2 cell differentiation. To examine this possibility, EMSAs were performed to assess changes in the E2F binding complexes. Differentiation of Caco-2 cells resulted in a dramatic increase of a slower-migrating E2F complex; supershift studies identified p130 as the major component of this complex. The increase of p130-E2F complexes with differentiation noted by supershift studies was further confirmed by DOC release EMSAs, showing an increase of dissociated E2F activity with Caco-2 postconfluence. Similar to our results in differentiated Caco-2 cells, this increase of p130-E2F complexes has been reported in other cell systems. Notably, the increase of p130-E2F complexes is associated with cell cycle block in hematopoietic cells and the terminal differentiation of myoblasts to myotubules and 3T3-L1 fibroblasts to adipocytes and mezerin-induced differentiation of human melanoma cells (7, 19, 23, 36, 37, 41, 43). Taken together, the shift of p130 from the cytoplasmic to the nuclear fractions and the induction of p130-E2F complexes further suggest the importance of the p130 protein in the initiation and/or maintenance of the terminally differentiated state in Caco-2 intestinal cells.

Similar to p130, we noted an increase of p107 nuclear protein levels associated with Caco-2 differentiation. These findings were somewhat surprising, since other studies have noted an actual decrease in p107 levels in quiescent or differentiated cell types in which p130-E2F was the preferential complex (23, 36, 43). However, consistent with our findings, induction of p107 levels has been noted in hexamethylene bisacetamide-induced differentiation of the murine erythroleukemia cell line (37). Collectively, these studies emphasize the apparent complexity of the mechanisms leading ultimately to cell cycle arrest and differentiation. In fact, p107 has been recently shown to mediate growth suppression by the inhibition of Cdk2-cyclin kinases in a manner independent of E2F (48). Moreover, in preliminary findings, we noted increased binding of p107 to Cdk2 at 3 days postconfluency, suggesting that p107 may contribute to the inhibition of Cdk activity noted in Caco-2 cells (unpublished data). Future studies are required to better ascertain the potential role of p107 in the suppression of Cdk activity in Caco-2 cells.

Despite increased p107 protein levels in the nuclear fractions with Caco-2 cell differentiation, we failed to detect binding of p107 to E2F in undifferentiated (preconfluent) or differentiated (postconfluent) Caco-2 cells, suggesting that p107 may only bind a small fraction of E2F in Caco-2 cells. This notion was further supported by the EMSAs demonstrating a decrease in dissociated E2F activity released by DOC from anti-p107 immune complexes in postconfluent Caco-2 cells. Although p107, independent of E2F, may play a role in Cdk suppression, our findings would suggest that the interaction of p107 with E2F members does not significantly participate in the differentiation process noted in postconfluent Caco-2 cells. No pRb-E2F complexes were detected in Caco-2 cells (either preconfluent or postconfluent) by EMSA. These findings were further corroborated by the DOC release EMSAs, which detected no "free" E2F activity in the pRb immune complexes. Therefore, these findings suggest that pRb does not interact with E2F in Caco-2 cells, despite the fact that pRb and the E2F family members that interact with pRb were identified in these cells.

In addition to regulation of E2F function through association with pRb or its related proteins, E2F can also be regulated by a change in protein levels and/or alterations of its phosphorylation state (9, 14, 41, 49). With Caco-2 differentiation, we found a marked decrease in E2F-4 phosphorylation; however, actual E2F protein levels were unchanged. These findings suggest that at least two mechanisms, which include p130 binding and differential phosphorylation, may regulate E2F activity during intestinal cell differentiation.

In conclusion, our findings indicate that Caco-2 cell cycle arrest and differentiation are associated with the predominant formation of p130-E2F complexes, suggesting that, as noted in other cell types, this complex is indicative of cell cycle exit. Another potential mechanism that may further serve to regulate E2F function in Caco-2 cells is differential E2F phosphorylation. Taken together with our previous findings (8, 12), the differentiation process in the Caco-2 intestinal cell line is not dependent on a single cellular mechanism, but, conversely, multiple components appear to contribute to the cell cycle block and ultimate maintenance of the differentiated phenotype. These multiple mechanisms include regulation of E2F function, induction of p130 and p107, downregulation of cyclin and Cdk protein expression, suppression of Cdk2 and Cdk4 activities, and induction of p21Waf1/Cip1. Although the Caco-2 cell line has been utilized by a number of investigators to assess potential mechanisms of enterocyte differentiation, it must be remembered that this is a transformed cell line. Therefore, even though this represents one of the better in vitro models of intestinal cell differentiation, the findings using Caco-2 cells may not be entirely applicable in vivo. The overall role of these pathways in the initiation and/or maintenance of the terminally differentiated state in intestinal cells remains to be fully elucidated.


    ACKNOWLEDGEMENTS

We are grateful to Dr. E. Aubrey Thompson for helpful suggestions and review of the manuscript. In addition, we thank Eileen Figueroa and Karen Martin for manuscript preparation.


    FOOTNOTES

This work was supported by National Institutes of Health Grants RO1 DK-48498, RO1 AG-10885, and PO1 DK-35608.

Q. Ding was a visiting scientist from the Institute of Radiation Medicine, Beijing, People's Republic of China. Z. Dong was a visiting scientist from the Institute of Biotechnology, Beijing, People's Republic of China.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: B. M. Evers, Dept. of Surgery, The University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555 (E-mail: mevers{at}utmb.edu).

Received 10 March 1999; accepted in final form 28 September 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 278(1):C110-C117
0363-6143/00 $5.00 Copyright © 2000 the American Physiological Society




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