p27Kip1 is an inducer of intestinal epithelial cell differentiation

Andrea Quaroni1, Jean Q. Tian2, Prem Seth3, and Colette Ap Rhys4

1 Section of Physiology, Cornell University, Ithaca, New York 14853; 2 Fort Dodge Laboratories, Fort Dodge, Iowa 50501-0518; 3 Human Gene Therapy Research Institute, Des Moines, Iowa 50309; and 4 Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Constant renewal of the intestinal epithelium is a highly coordinated process that has been subject to intense investigation, but its regulatory mechanisms are still essentially unknown. In this study, we have demonstrated that forced expression of the cyclin-dependent kinase inhibitors (CKIs) p27Kip1 and p21Cip1/WAF1 in human intestinal epithelial cells led to expression of differentiation markers at both the mRNA and protein levels. Cell differentiation was temporally dissociated from inhibition of retinoblastoma protein phosphorylation and growth arrest, already established 1 day after infection with recombinant adenoviruses. p27Kip1 proved significantly more efficient than p21Cip1/WAF1 in induction of cell differentiation. In contrast, forced expression of p16INK4a resulted in growth arrest without induction of differentiation markers. These results implicate both p27Kip1 and p21Cip1/WAF1 in the differentiation-timing process, but p21Cip1/WAF1 may act indirectly by increasing p27Kip1 levels. These results also suggest that induction of intestinal epithelial cell differentiation by CKIs is not related to their effects on the cell cycle and may involve interactions with cellular components other than cyclins and cyclin-dependent kinases.

cyclin-dependent kinase inhibitors; p21Cip1/WAF1; small intestine


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

AFTER BIRTH THE DIGESTIVE and protective functions of the intestinal mucosa depend on constant renewal and differentiation of the surface epithelium. This process starts with a small number of pluripotent stem cells, or a single "master" stem cell with a cohort of "daughter" cells, all located in the lower five cell positions in the crypts (8, 37, 51, 60). The intestinal stem cells cannot be identified with certainty due to the lack of known markers, but they are assumed to have an enormous proliferative potential (51). With each cell division, they can either renew themselves or become "committed" crypt cells that undergo a limited number of rapid divisions before exiting the cell cycle and differentiating. The entire process from cell generation in the crypts to loss in the upper regions of the villi takes 3-5 days, depending on animal species and stage of development (4, 6). Incisive data regarding stem cell characteristics and gene regulation in crypt cells have come from studies centered on chimeric (60) and transgenic (22) animals, but the basic mechanisms responsible for induction of cell differentiation are still little understood. The decision to embark on such an irreversible pathway is taken by the committed crypt cells abruptly, while in their most rapid state of proliferation (6). The newly differentiated cells acquire their distinctive ultrastructural features and cell surface markers after leaving the proliferative cell cycle, at the top of the crypts or base of the villi (15, 52, 56). In the region of the crypts where the committed epithelial cells lose their proliferative potential, there are no known landmarks that can be clearly identified as determinants of cell differentiation. This is also highlighted by the considerable flexibility of the overall process, enabling it to respond and adapt very well to changes in mucosal needs. For example, when an increase in cell production is required (i.e., to compensate for abnormal loss of cells on the villi), it is achieved by displacement of the proliferative cut-off region toward the mouth of the crypts or even the lower third of the villi, markedly increasing the size of the proliferative cell population (59). Thus differences in basement membrane composition along the crypt-villus axis (2, 3, 31), unquestionably important in modulating gene expression and in directing cell migration, are not likely to represent the only stimulus for differentiation. An alternative hypothesis is that the process is driven by an intracellular molecular "clock" that activates when stem cells become committed transit cells, flexible enough to accommodate a limited variability in the number of total cell divisions under the influence of physiological extracellular stimuli, but inexorable in the final outcome.

Among the most attractive candidate components of such a clock are the cyclin-dependent kinase inhibitors (CKIs) p27Kip1 (p27) and p21Cip1/WAF1 (p21). An important characteristic of these proteins is that they function in a stoichiometric rather than catalytic fashion (50, 58, 70); thus a gradual increase in their concentration after the crypt cells leave the stem cell compartment could allow a limited number of cell divisions to take place before achieving a critical threshold, interpreted by the crypt cells as a stimulus for differentiation. Soon after p21's initial discovery as a molecule induced by wild type but not mutant p53 (16), which was markedly increased in expression in senescent cells (41) and capable of associating with most cyclin-dependent kinase (Cdk) complexes (25), it became apparent that its expression pattern correlated with terminal differentiation of multiple cell lineages in vivo (43) and in tissue culture cells (23, 29, 63). There are, however, conflicting reports (26), and forced expression of p21 in terminally differentiated primary keratinocytes actually downregulated markers of differentiation (13). In the intestinal tract, the highest levels of p21 mRNA were observed by in situ hybridization in differentiated enterocytes (43), but somewhat different results were obtained by protein immunostaining (13, 53). In the colorectal epithelium, its expression pattern appeared to correlate with loss of proliferation (17, 46), a conclusion consistent with studies conducted on cultured human colon cell lines (1, 14, 33) where p21 was found to represent a critical effector of butyrate-induced growth arrest (1). More recently, p27 has also been implicated in cell differentiation: in keratinocytes, its level was found to increase when cells were induced to differentiate, and pretreatment of adherent cells with p27 antisense oligonucleotides prevented the onset of differentiation (27).

In a previous study, by using the conditionally immortalized human intestinal epithelial cell line tsFHI (65), we have demonstrated that growth arrest and cell differentiation led to induction of both p21 and p27, but with markedly different kinetics. The p21 increase was rapid but transient, coinciding with the onset of growth arrest, whereas that of p27 was delayed and sustained, preceding the expression of differentiation markers by about 1 day. Surprisingly, in spite of its more than fivefold increase in differentiating cells, p27 failed to complex in significant amounts with any of the cyclins and Cdks detected in tsFHI cells. We have interpreted these findings to indicate that p21 is the main CKI involved in irreversible growth arrest during the early stages of crypt cell differentiation in association with D-type cyclins, cyclin E, and Cdk2, whereas p27 may induce or stabilize expression of differentiated traits that act independently of cyclin-Cdk function.

In this study, we sought to obtain direct evidence for the ability of these CKIs to induce intestinal cell differentiation when forcibly overexpressed in the normal human intestinal epithelial cell line HIEC6 (45). We demonstrate here that expression of both p21 and p27, but not of the unrelated CKI p16INK4a (p16), resulted in cell differentiation. Further, the temporal dissociation between growth arrest and appearance of differentiated markers indicated that these CKIs are likely to act independently from their ability to complex with cyclins and Cdks that are primary regulators of cell cycle progression during the G1 phase.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture and adenovirus infection. The normal fetal human intestinal epithelial cell line HIEC6 was established and characterized by Perreault and Beaulieu (45). In this study, it was used between passages 7 and 12, and routinely cultured in OptiMEM I (GIBCO BRL-Life Technologies, Grand Island, NY) supplemented with 4% fetal bovine serum, 2 mM glutamine, 2 mM L-alanyl-glutamine (GlutaMAX I, GIBCO BRL-Life Technologies), 10 mM HEPES, 10 ng/ml epidermal growth factor (human recombinant EGF; Upstate Biotechnology, Lake Placid, NY), 50 U/ml penicillin, 50 µg/ml streptomycin at 37°C, and 6% CO2. In some experiments, cells were cultured on extracellular matrix proteins (fibronectin, laminin, E-C-L) or with hormones [transforming growth factor-beta (TGF-beta ), EGF, thyroxine, dexamethasone] as described for tsFHI cells (53). Cells were processed for scanning electron microscopy as previously described (55). Before infection with adenovirus, cells were plated and cultured in standard medium for 48 h. At the time of infection, one plate was trypsinized and counted, then the others were supplemented with 0.5 ml (35-mm diameter dishes) or 3 ml (100-mm dishes) of standard culture medium that contained adenovirus at the desired multiplicity of infection. The plates were returned to the incubator and kept with occasional rocking for 90 min. Thereafter, 1.5 ml or 7 ml, respectively, of complete media were added to each plate, and the cells were further incubated for the duration of the experiment. Control plates were subjected to the same procedure, but no adenovirus was added. The recombinant adenoviruses used in this study have been described previously (9, 10, 30). N-acetyl-leucinyl-leucinyl-norleucinal was obtained from Sigma (St Louis, MO) and added to the culture medium at 2, 5, and 10 µM concentrations; control cultures received the solvent dimethyl sulfoxide alone.

Flow cytometry. For flow cytometry, cells were harvested by trypsinization, washed with phosphate-buffered saline (PBS), and then resuspended in 1 ml PBS. The cells were fixed by addition of 2 ml of 100% methanol and incubation on ice for 30 min. Finally, they were spun down and stained with propidium iodide (PI) for 30 min (11). Cell cycle analysis was performed on a Becton Dickinson FACScan using Cellquest software for data acquisition and ModiFit software for analysis. For each sample, 20,000 cells were registered, and >10,000 registered events were gated by PI (FL2-A and FL2-W). All data shown represent only the gated cells.

DNA synthesis. DNA synthesis was determined in two ways. First, we measured the incorporation of [methyl-3H]thymidine into cellular DNA. Cells were grown in 60-mm dishes; the medium was removed, the cells were washed twice with standard complete medium, then fresh medium that contained 0.5 µCi/ml [methyl-3H]thymidine (20 Ci/mmol; NEN Products, Boston, MA) was added. After a 1-h incubation at 37°C, the medium was aspirated, and each dish was sequentially washed twice with PBS and treated twice with cold 5% TCA (for 10 min each time). The cells were then dissolved in 1 ml 0.1 N NaOH, and aliquots of the solution were used for determination of the radioactivity incorporated (by scintillation counting) and of the protein content [by the method of Lowry et al. (38)]. Second, DNA synthesis was assessed by using the bromodeoxyuridine (BrDU) labeling and detection kit from Boehringer Mannheim (Indianapolis, IN), following the manufacturer's protocol. Briefly, cells were incubated for 2 h with culture medium that contained 10 µM BrDU, and then cells were washed with PBS, fixed with acidic ethanol, and incubated with anti-BrDU antibody in the presence of nucleases for DNA denaturation. BrDU that was incorporated into cellular DNA was visualized by fluorescence microscopy.

Immunofluorescence staining. Cultured cells were washed three times with PBS, fixed with 3% formaldehyde, and then either directly processed for immunofluorescence staining [nonpermeabilized samples, staining for dipeptidylpeptidase IV (DPPIV) and aminopeptidase N (APN)] or permeabilized by one of two methods: 1) incubation with acetone:methanol 1:1 at -20°C for 10 min or 2) lysis with 0.2% Triton X-100 in PBS for 2 min at room temperature. Further processing of the samples was as previously described (65). The secondary antibodies were FITC- or rhodamine-conjugated goat anti-mouse or donkey anti-rabbit IgG, obtained from Boehringer Mannheim, and diluted 1:25 in PBS. Cells were counterstained with 0.01% Evans blue for 2 min. After antibody incubations and washings, the cells were mounted in glycerol:PBS (9:1) + 2.5% 1,4-diazabicyclo[2.2.2]octane and covered with coverslips. Stained cells were examined with a Zeiss Axiovert 10 microscope equipped with epifluorescence optics and an Optronics 3 chip charge-coupled device camera. Digital images were processed with Adobe Photoshop software.

Enzyme assays. DPPIV and APN activities were determined as previously described (53). Cyclin D-associated kinase activity was assayed as described for tsFHI immunoprecipitates (65).

Ribonuclease protection assay. Total cellular RNA was isolated from cultured cells with the use of an RNeasy kit from Qiagen (Chatsworth, CA). The integrity of the RNA was verified by ethidium bromide staining, and the quantity was determined spectrophotometrically. DPPIV and APN mRNA levels were evaluated using a ribonuclease protection assay described previously (66), normalized with respect to 18S ribosomal RNA levels in each sample.

Immunoblotting. Total cell lysates solubilized in SDS-PAGE sample buffer were subjected to SDS-PAGE and Western blotting, performed essentially as described previously (65). Three different gels were used for separating proteins: 7.5% acrylamide for retinoblastoma protein (pRb) and DPPIV; 15% for p16; and 12% for p21 and p27. After electrophoresis, proteins were electroblotted onto nitrocellulose membranes (Hybond nitrocellulose; Amersham) using a transblot system (Bio-Rad) at 100 V, 5°C for 90 min. The membranes were blocked at 4°C overnight in blocking buffer that contained PBS, 0.1% Tween 20, and 3% bovine serum albumin, incubated with primary antibody diluted in blocking buffer at room temperature for 2 h, and washed three times in washing buffer that contained PBS and 0.1% Tween 20. Appropriate secondary antibodies (1:3,000 dilution of either horseradish peroxidase-linked sheep anti-mouse immunoglobulin or donkey anti-rabbit immunoglobulin; Amersham) were incubated with membranes for 1 h at room temperature. Specific proteins were detected with the use of an enhanced chemiluminescence system (ECL protocol, Amersham Life Science). The intensities of protein bands were analyzed with the use of an LKB Ultroscan densitometer (LKB, Bromma, Sweden).

Sources of antibodies. The monoclonal anti-human DPPIV and APN antibodies used in this study have been previously produced and characterized in our laboratory (57). The following affinity-purified polyclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and used for immunofluorescence staining and for Western blotting: rabbit-anti-p16 (C-20) [sc-468] against a peptide corresponding to amino acids 137-156 mapping at the carboxy terminus of the human p16; rabbit-anti-p21 (C-19) [sc-397] against a peptide corresponding to amino acids 146-164 mapping at the carboxy terminus of the human p21; rabbit-anti-p27 (C-19) [sc-528] against a peptide corresponding to amino acids 181-198 at the carboxy terminus of the human p27; rabbit-anti-pRb (C-15) [sc-50] against a peptide corresponding to amino acids 914-928 mapping at the carboxy terminus of the human pRb p110; a mouse monoclonal against the full length () human cyclin D1; rabbit-anti-cyclin D2 (C-17) [sc-181] against a peptide corresponding to amino acids 274-289 mapping at the carboxy terminus of human cyclin D2; and rabbit-anti-cyclin D3 (C-16) [sc-182] against a peptide corresponding to amino acids 277-292 mapping at the carboxy terminus of the human cyclin D3. Donkey anti-rabbit or sheep anti-mouse antibodies conjugated with horseradish peroxidase were purchased from Amersham Life Science (Arlington Heights, IL).

Statistical analysis. Analysis of variance was used as a statistical test. Results are expressed as means ± SE and were considered significant when P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

HIEC6 is a normal epithelial cell line, established from human fetal small intestinal crypts (45), that does not express significant amounts of any of the differentiation markers we have tested (APN, DPPIV, sucrase-isomaltase, lactase, alkaline phosphatase, and villin). Rare, widely scattered DPPIV-positive cells are occasionally observed (see Fig. 6), but their frequency never exceeded 0.1% in our experiments. By immunofluorescence staining, neither p16 nor p27 could be detected in these cells, either subconfluent (not shown) or confluent for 1-7 days (Fig. 1B). Although a majority of the cells was also p21 negative, a significant fraction (5.3-8.7% in 5 separate samples) displayed nuclei intensely positive for p21 (Fig. 1B). By Western blotting that used total cell lysates, p27 was undetectable, whereas weak bands stained with p16 and p21 antibodies were observed (their intensity was essentially identical to that shown in Fig. 1A for Adnull-infected cells). Our initial attempts to induce HIEC6 cell differentiation included culture in the presence of 1 ng/ml TGF-beta or on plastic dishes coated with laminin, type I collagen, or E-C-L basement membrane. These treatments have been reported to induce at least limited differentiation in some rat intestinal epithelial cell lines (7, 32). All our attempts failed to induce expression of DPPIV or APN, markers of differentiation most commonly expressed in differentiated intestinal cells in vitro (44, 53). TGF-beta was of particular interest because, in some cell lines, it has been found to promote G1 arrest by inhibiting cyclin-Cdk kinases through the cooperative action of p21 or p27 and of an Ink4 family member (58, 68). Treatment of HIEC6 cells for 24 or 48 h with 1 or 2 ng/ml TGF-beta did produce a marked inhibition of cell proliferation (78.2 ±9.8%, n = 6, as estimated by the [3H]thymidine incorporation method; 73.5 ±5.2%, n = 10 by the BrDU method). However, in TGF-beta -treated cells, we did not observe any significant induction of p27 or DPPIV or increase in the fraction of p21-positive nuclei. These findings are quite similar to those obtained using tsFHI cells, in which TGF-beta also produced growth inhibition without increasing p21 or p27 levels (65).


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Fig. 1.   Native and forced expression of cyclin-dependent kinase inhibitors (CKIs) in HIEC6 cells. A: Western blot analysis. HIEC6 cells were infected with recombinant adenoviruses (or Adnull, an adenovirus with no exogenous cDNA used as control) at a multiplicity of infection of 10-100 plaque-forming units (pfu)/cell and analyzed 1 day later. Total cell lysates (50 µg protein/sample) were resolved by 12.5% SDS-PAGE and immunoblotted with antibodies specific for p16, p21, or p27. B: immunofluorescent staining for p16, p21, or p27. HIEC6 cells, uninfected (control), or infected with recombinant adenoviruses at a multiplicity of infection of 100 pfu/cell, were fixed and stained 1 day after infection. In other experiments, we verified that CKI expression in infected cells was maintained at levels similar to those shown here for 2-7 days after infection.

Because the intracellular concentrations of p27 and p21 are, in many cell types, under posttranscriptional regulation via the ubiquitin-proteasome system (5, 13, 39), we have tried to increase their expression by incubating HIEC6 cells for 24 or 48 h with the proteasome inhibitor N-acetyl-leucinyl-leucinyl-norleucinal (42) at 2-, 5-, or 10-µM concentrations. None of these treatments significantly increased the frequency of p21-positive cells or induced p27 expression. A 1.5- to 2-fold increase in p21 was observed by Western blotting using total cell lysates, indicating that its level was slightly higher in cells that already expressed this CKI. Immunofluorescence staining and Western blotting demonstrated that neither DPPIV nor APN were induced. Overall, these experiments indicated that expression of p21 and p27 in HIEC6 cells is dependent on specific stimuli that were not provided by the treatments described above.

Adenovirus-mediated expression of CKIs. To force expression of p16, p21, and p27 in HIEC6 cells, we infected them with adenovirus constructs that contained the corresponding cDNAs (Adp16, Adp21, and Adp27) under the control of the murine cytomegalovirus immediate early gene promoter. An adenovirus that lacked any exogenous cDNA (Adnull), or uninfected cells, were used as negative controls in these experiments. We have chosen adenoviral vectors (rendered replication incompetent by deletion of E1 sequences) over other methods of transfection because they have been reported to infect a variety of cultured human epithelial cells with nearly 100% efficiency (9, 10, 30, 61). Because the adenoviruses we have used differ only in their respective cDNA sequences, there should be no major differences in either the levels of transgene expression or infectivity of the recombinant virus particles.

Efficient expression of the three CKIs in HIEC6 cells 24 h after infection with 10-100 plaque-forming units (pfu)/cell was verified by Western blotting and by immunofluorescence staining (Fig. 1). At the protein level, Western blot analysis demonstrated that all three CKIs were expressed in amounts increasing with the viral load. The presence of even the highest levels of p16 did not influence expression of the other two CKIs, whereas in Adp21-infected cells, there was a significant induction of p27, particularly in cells infected at 100 pfu/cell. Adp27 did not alter p21 expression but slightly increased p16 levels. Immunofluorescence staining of cells infected at 100 pfu/cell confirmed and extended these results (Fig. 1B): 73-84% (range of 5 experiments) of cells infected with Adp16 or Adp27 were intensely stained with antibodies to p16 or p27, respectively; fluorescence was most intense over the nuclei, but included the entire cytoplasm. In Adp21-infected cells, 97-100% of the cells were positive for p21, with the fluorescence confined to their nuclei. Similar results were obtained at 50 pfu/cell (not shown), with only the frequency of p16-positive cells being slightly lower (61-74% in 3 experiments). No CKI induction was observed in Adnull-infected cells. In separate experiments, we verified that CKI expression in infected cells was maintained at similar levels for at least 7 days after infection (not shown).

Inhibition of cell proliferation by CKIs. Forced expression of all three CKIs resulted in essentially complete growth arrest in HIEC6 cells 24 h after infection. In cells infected with 100 pfu/cell, incorporation of radioactive thymidine into DNA was reduced by >95% (Fig. 2A). Inhibition was already significant in cells infected with 1 pfu/cell, and its extent correlated with viral loads, as illustrated using the BrDU method (Fig. 2B). At each viral load within the range of 1-50 pfu/cell, inhibition of DNA synthesis was significantly greater in Adp27-infected cells, followed by Adp21 and Adp16, but in all cases, <3% of the cells infected with 100 pfu/cell incorporated BrDU into their nuclei. DNA synthesis was not significantly altered in Adnull-infected cells (the slight increase apparent in Fig. 2A was not significant and not observed in all experiments). In cells infected with Adp16, Adp21, or Adp27 (at a multiplicity of infection of 50 or greater), S phase cells were essentially undetectable (<3% of the nuclei were stained by the BrDU-incorporation method) up to 7 days after infection (later times were not examined, see Fig. 2C for Adp27-infected cells; identical results were obtained with cells infected with Adp16 at 100 pfu/cell or Adp21 at 50 pfu/cell).


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Fig. 2.   Inhibition of HIEC6 cell proliferation by exogeneous CKIs. Cells uninfected (control), infected with adenovirus with no exogenous cDNA (Adnull), or with recombinant adenoviruses (Adp16, Adp21, Adp27) were analyzed 1-7 days after infection. A: DNA synthesis assayed 1 day after infection (at 100 pfu/cell) with a 1-h pulse of [3H]thymidine; values are expressed as percentages of [3H]thymidine incorporated into DNA compared with uninfected controls (n = 5; error bars indicate SD). B: dose dependence of inhibition of cell proliferation. Cells, infected at 1-100 pfu/cell, were assayed after 1 day by the bromodeoxyuridine (BrDU) incorporation method. C: persistent inhibition of cell proliferation up to 7 days after infection with recombinant adenoviruses; in this experiment, cells infected with Adp27 at 50 pfu/cell, or uninfected controls, were assayed daily by the BrDU incorporation method. Identical results were obtained with cells infected with Adp16 (at 100 pfu/cell) or Adp21 (at 50 pfu/cell). D: inhibition of retinoblastoma protein (pRb) phosphorylation 1 day after infection. Total cell lysates were subjected to SDS-PAGE and immunoblotted with a pRb-specific antibody. The hyperphosphorylated pRb appears as a lower mobility band whose intensity is unaffected by infection with Adnull, is markedly reduced (>90%) in Adp16-infected cells, and is essentially absent in p21- and p27-infected cells. E: cyclin D-associated kinase activity measured, 1 day after infection, in immunoprecipitates (cyclins D1, D2, and D3 were precipitated together) from cells infected at 100 pfu/cell.

Control and adenovirus-infected cells were also analyzed by flow cytometry. Results from a representative experiment in which cells were infected at 100 pfu/cell are shown in Fig. 3. They illustrate that, in CKI-overexpressing cells, there was a reduction in the fraction of S phase cells to negligible levels (<1%), with correspondent increases in the fraction of G1/G0 cells. Essentially identical results were obtained with cells infected at 50 pfu/cell. Adnull, at the doses used in this study (10-100 pfu/cell), produced no significant effects on DNA histograms, but at much higher doses (500 pfu/cell or greater) it decreased the S phase cells by 42% (average of 4 experiments).


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Fig. 3.   CKI-overexpressing HIEC6 cells do not enter the S phase of the cell cycle. Cells, infected at 100 pfu/cell, were analyzed by flow cytometry. Forced expression of all 3 CKIs reduced the fraction of cells in S phase to negligible levels, with corresponding increases in G1 (or G0) cells.

Block of cells at the G1/S interphase by p16, p21, and p27 normally involves inhibition of Cdks responsible for phosphorylation of the pRb protein. This was also the case for infected HIEC6 cells. After 24 h of infection, the lower mobility hyperphosphorylated pRb band was essentially undetectable in cells infected with Adp21 or Adp27; in Adp16-infected cells, inhibition of pRb phosphorylation was marked (>85% by densitometric analysis) but not as complete (Fig. 2D). A corresponding decline in the kinase activity associated with cyclin D immunoprecipitates (cyclins D1, D2, and D3 were coprecipitated in these experiments) was also observed (Fig. 2E). The residual kinase activity observed in Adp16-infected cells may have resulted from Cdk2-containing complexes, but it apparently was not sufficient to drive cells into the S phase.

Induction of cell differentiation. Forced overexpression of p27 produced marked morphological changes in HIEC6 cells, most notably a two- to threefold increase in cell volume (estimated by flow cytometry), acquisition of a polygonal cell shape, and a dramatic increase in the frequency of apical microvilli (Fig. 4). Adp16-infected cells were also slightly larger (1.5-fold volume increase on average) and more polygonal in shape than control cells (Fig. 4), but the frequency of apical microvilli was not significantly increased. Adp21-infected cells consisted of a mixed population, with about 20-40% of the cells (in 3 separate experiments) resembling p27-infected cultures, the rest resembling Adp16-infected cells (not shown).


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Fig. 4.   Morphology of control and infected HIEC6 cells. Cells were examined by scanning electron microscopy 7 days after infection with Adp16 or Adp27 at 50 or 100 pfu/cell (producing essentially identical results). Note the much larger surface area of infected cells and the presence of a dense layer of microvilli covering the apical surface of Adp27-infected cells. Adp16-infected cells were also moderately increased in surface area and displayed a more polygonal shape than control cells, but lacked the dense mat of surface microvilli.

After infection with Adp27 at 50 or 100 pfu/cell (producing essentially identical results), DPPIV expression at both the protein (Fig. 5A) and mRNA (Fig. 5B) levels remained very low or negligible during the first 3 days after infection. A significant increase in both mRNA and protein was observed by days 4 or 5, but maximal induction was only achieved 6 and 7 days after infection. In contrast, p27 protein levels were consistently high and did not vary significantly between days 2 and 7 after infection (Fig. 5A). Similar results were obtained using Adp21, which was also able to induce DPPIV expression, but at significantly lower (3- to 4-fold) levels than p27 (not shown). In contrast, no induction of DPPIV was observed at any time in cells infected with Adnull or Adp16 up to 200 pfu/cell (not shown). DPPIV expression was also verified by measuring its enzyme activity in cell lysates (Fig. 5C): 7 days after infection with Adp21 or Adp27 at 100 pfu/cell, there was a significant increase in enzyme activity that was 2.5 to 3 times higher in Adp27- than in Adp21-infected cells.


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Fig. 5.   Induction of dipeptidylpeptidase IV (DPPIV) expression by p21 and p27. A: Western blot analysis of DPPIV in HIEC6 cells infected with Adp27. Cells were infected at 100 pfu/cell and analyzed 2-7 days after infection. Control cells were uninfected. During the same period, p27 levels in Adp27-infected cells (detected by blotting as in Fig. 1) did not vary significantly. A similar pattern of DPPIV protein induction was observed for cells infected with Adp21, but band intensity was 2-3 times lower at 6 and 7 days. B: DPPIV mRNA levels evaluated by ribonuclease protection assay. Cells were either uninfected (control) or infected with Adp27 at 100 pfu/cell. In each assay, 40 µg of total RNA were probed, and a human 18S ribosomal probe was used as internal control to normalize RNA loads. C: DPPIV enzyme activity in uninfected HIEC6 cells (control), cells infected with Adnull, and cells infected with recombinant adenoviruses (at 100 pfu/cell). Cells were analyzed 7 days after infection in all cases. *Significant difference (P < 0.05) from control and Adnull-infected cells; **significant difference (P < 0.05) compared with both control and Adp21-infected cells.

Immunofluorescence staining of cells 6 days after infection confirmed and extended these results (Fig. 6): Adnull- and Adp16-infected cells contained a very small fraction (<1%) of DPPIV-positive cells, not significantly different from that in control (uninfected) cells. In contrast, DPPIV-positive cells were far more frequent in Adp21- (25 ±6%) and Adp27- (73 ±12%) infected cells. In the latter case, their frequency approached that of p27-expressing cells (Fig. 1).


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Fig. 6.   Immunofluorescence staining for DPPIV of HIEC6 cells uninfected (control), infected with Adnull, or with recombinant adenoviruses at 100 pfu/cell. Cells were fixed and stained 6 days after infection. The red color corresponds to the Evans blue counterstain. To determine the fraction of DPPIV-positive cells, 5 fields of at least 200 cells each were counted for each sample. *Significant difference (P < 0.05) from control and Adnull-infected cells; **significant difference (P < 0.05) compared with both control and Adp21-infected cells.

Expression of another marker of intestinal cell differentiation, APN, followed similar or identical kinetics. APN mRNA was essentially undetectable in control cells and in cells infected with all three recombinant adenoviruses during the first 2-3 days after infection (Fig. 7A). In cells infected with Adp27 at 50 or 100 pfu/cell, a significant increase in APN mRNA was observed by 5 days, but maximal expression was achieved 6-7 days after infection (at 7 days in the experiment shown in Fig. 7A). APN enzyme activity in cells examined 7 days after infection with the different adenovirus constructs was significantly increased only in Adp21- and Adp27-infected cells, the latter displaying by far the highest APN-specific activity (Fig. 7B). Very similar results were obtained with cells stained for APN (Fig. 8): control, Adnull-, and Adp16-infected cells were essentially negative. A marked induction in APN expression was observed in both Adp21- and Adp27-infected cells, with Adp27 again significantly more effective. Another marker of differentiation in HIEC6, enkephalinase (EC 3.4.24.11), was also induced in Adp21- and Adp27-infected cells, with a pattern nearly identical to that of APN (data not shown).


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Fig. 7.   Induction of aminopeptidase N (APN) expression. A: APN mRNA levels, evaluated by ribonuclease protection assay, in control and in cells infected with Adp27 at 100 pfu/cell. In this assay, 40 µg of total RNA were probed, and a human 18S ribosomal probe was used as internal control to normalize RNA loads. B: APN enzyme activity in uninfected HIEC6 cells (control), cells infected with Adnull, and cells infected with recombinant adenoviruses (at 100 pfu/cell). Cells were analyzed 7 days after infection. *Significant difference (P < 0.05) from control and Adnull-infected cells; **significant difference (P < 0.05) compared with both control and Adp21-infected cells.



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Fig. 8.   Induction of APN expression by p21 and p27. HIEC6 cells were infected at 100 pfu/cell and analyzed by immunofluorescence staining 6 days after infection. The red color corresponds to the Evans blue counterstain. To determine the fraction of APN-positive cells, 5 fields of at least 200 cells each were counted for each sample. *Significant difference (P < 0.05) from control and Adnull-infected cells; **significant difference (P < 0.05) compared with both control and Adp21-infected cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To the best of our knowledge, this study represents the first demonstration of the ability of p27 to promote differentiation of an epithelial cell type. Since its initial discovery as an inhibitor of Cdk-cyclin complexes with broad target specificity (47, 48, 68), a variety of other functions have been attributed to this CKI, including tumor suppression and induction of apoptosis, cell cycle exit, and cell differentiation (reviewed in Ref. 25). The latter was inferred mainly from its increased expression in a variety of terminally differentiated cells (26, 27) but has been difficult to demonstrate conclusively. For example, in cultured epidermal keratinocytes, the timing of the upregulation of p27 correlated closely with induction of squamous-specific genes, and treatment with p27 antisense oligonucleotides prevented the onset of differentiation. Inhibition of Cdks and growth arrest were not affected by the oligonucleotides, linking p27 specifically with the differentiation program (27). However, forced expression of p27 (and of p16 or p21) by infection with recombinant adenoviruses proved insufficient to induce expression of the differentiation genes, leading the authors to conclude that other signals provided by suspension culture are necessary to trigger terminal squamous differentiation. This was clearly not the case in HIEC6 cells infected with Adp27, and the difference may lie in the intrinsic properties of these two epithelia. In vivo, epidermal keratinocytes form a stratified epithelium in which growth arrest and differentiation start with detachment from the basal layer. On the other hand, intestinal epithelial cells carry out their entire renewal process while attached to a basement membrane and thus should not be expected to require additional signals generated by cell suspension.

Given the wide variety of differentiated cell types, it is not surprising that common signaling molecules are used in different ways to achieve the same final outcome. Thus in the myelomonocytic U-937 cell line forced to transiently overexpress p21 and/or p27 by plasmids carrying the corresponding cDNAs, a G1 arrest consequent to inhibition of cyclins and Cdks by p21 or p27 was found to be sufficient to activate the differentiation pathway (34). In contrast, in HIEC6 cells infected with Adp27 (or Adp21), there was a clear dissociation between growth arrest (established already after 1 day) and expression of DPPIV or APN 3-4 days later. Further evidence that growth inhibition alone was not sufficient to start the differentiation process was provided by cells induced to express p16. These findings correlate well with those of our previous study on tsFHI cells: irreversible growth arrest caused by treatment with differentiation medium or TGF-beta at the permissive temperature failed to induce cell differentiation, and a similar lag period of 2-4 days was observed between growth inhibition at the nonpermissive temperature and expression of DPPIV and APN (65). This parallelism also indicates that differentiation of these two intestinal cell lines may be driven by essentially the same mechanisms, in spite of the presence of a SV40-T antigen in tsFHI cells.

Infection of HIEC6 cells with Adp21 was also found to promote expression of DPPIV and APN, but it is unclear whether this was a direct effect, because p27 was also induced 1 day after infection (Fig. 1A). On the contrary, most evidence coming from our studies on HIEC6 and tsFHI cells, and from analysis of the p21 expression pattern in the intestine, seems to argue against a direct involvement of this CKI in induction of differentiation markers in this epithelium. In tsFHI cells cultured at the nonpermissive temperature, p21 was upregulated rapidly (within 8 h) but started to decline well before expression of DPPIV or APN was observed (65). In uninfected HIEC6, p21-positive cells were uncommon (5.3-8.7%, see Fig. 1B) but still ~10 times more frequent than DPPIV-positive cells (<1%, see Fig. 6). Similarly, after Adp21 infection, there was no correlation between the frequency of p21-positive cells (>95%) and that of cells expressing DPPIV (25%) or APN (26%). A possible explanation for these findings is that in a fraction of Adp21-infected cells, there was sufficient inhibition of cyclin E-Cdk2 to increase p27 levels enough to promote differentiation. Such an interpretation is supported by the fact that, although in most cells types examined, p21 expression was found to be dependent on transcriptional activation, changes in p27 levels were attributed to increased mRNA translation or decreased protein degradation (28, 40, 62). Phosphorylation of p27 by cyclin E-Cdk2 is known to predispose this protein to intracellular degradation (62), and inhibition of this kinase activity was also a likely explanation for the upregulation of p27 in tsFHI cells (65).

The expression pattern of p21 in the intestine is also indicative of an involvement of this CKI in growth arrest rather than differentiation: p21 is induced in cells adjacent to the proliferative compartment (17, 46), or even somewhat lower (53), but downmodulated at later stages of differentiation (13, 53). p21 was also found to be a crucial effector of butyrate-induced growth arrest in colon tumor cell lines expressing small intestinal markers (1). A correlation between p21 expression and growth arrest, but not cell differentiation, has been noted in other epithelia (17, 20, 49), and in cultured keratinocytes, infection with Adp21 was actually found to inhibit expression of markers of terminal differentiation at both the protein and mRNA levels.

Possible mechanisms of action of p27 and p21. The ability of p16 to promote growth arrest but not differentiation of HIEC6 cells, and the extended lag period between inhibition of pRb phosphorylation/growth arrest and induction of DPPIV or APN in Adp27-infected cells, indicate that the differentiation-promoting activity of p27 was not simply due to its ability to inhibit G1 cyclin-Cdk complexes. This conclusion is consistent with our previous observation that, in tsFHI cells, after induction by a temperature shift, p27 did not significantly associate with any of the Cdks or cyclins detected in these cells (65). Because the p27 expressed by tsFHI cells was biochemically and immunologically indistinguishable from that expressed by other cell types, a likely explanation for this unusual observation is that, at the time p27 levels increased, most cyclins and Cdks present were already complexed with previously expressed p16 and p21 (the only other CKIs expressed in these cells). These considerations indicate that the mechanism of action of p27 in inducing expression of differentiation markers in intestinal cells may involve an interaction with yet to be identified cellular components. A possible candidate is Jab1, a protein found to interact with p27 using a yeast two-hybrid screen and shown to promote its movement from the nucleus to the cytoplasm and its subsequent degradation (67). It is noteworthy that Jab1 was not found to interact with p21.

A model for the role of CKIs in intestinal cell differentiation. Our working hypothesis concerning the role of CKIs in intestinal cell differentiation, highlighting the main results of this study, is presented in Fig. 9. We hypothesize that a key property distinguishing committed proliferative crypt cells from stem cells is their ability to express p21, and it is this ability that intrinsically limits their proliferative potential. Growth arrest could result, after a limited number of cell divisions, either from the progressive accumulation of p21 beyond a threshold level or progressive loss of factors temporarily inhibiting p21 expression at the protein level, for example, by promoting its degradation. These two mechanisms are not mutually exclusive, and either or both could sense and integrate extracellular stimuli coming from the mesenchyme, lamina propria, or even the proliferative cell compartment in a feedback inhibition of growth (59); all are factors known to influence crypt cell dynamics (reviewed in Ref. 54). The differentiation process would start with p21-driven irreversible growth arrest, accompanied by inhibition of cyclin E-Cdk2 or other kinases responsible for p27 degradation. A rise in p27 levels would ultimately activate, undoubtedly by interaction with a variety of other molecules, expression of differentiation genes. The process would end with apoptosis, known to be activated shortly before villus cells are shed into the lumen (21). Although this model attempts to organize our present knowledge, it certainly represents an oversimplification of the real process. Just in principle, it is highly unlikely that a cellular system constantly exposed to potential environmental carcinogens like the intestine could depend on a single gene for induction of its differentiation. It is far more likely that multiple redundant genes are involved in this process, as the normality of the intestinal tract in p21-deficient mice implies (12). It is of interest, however, that both p27 nullizygous and heterozygous mice were found to be predisposed to intestinal tumors (19), stressing the importance of this CKI in the intestinal epithelium.


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Fig. 9.   This diagram illustrates our working hypothesis concerning the function of p21 and p27 in growth regulation and differentiation of intestinal epithelial cells. It highlights 3 distinct stages in epithelial cell development, factors influencing committed cell dynamics, the presence of an intrinsic clock limiting proliferation of committed crypt cells, the role of p21 in irreversible cell growth arrest, and of p27 in induction of differentiation markers.

Implications for intestinal cancer. The demonstration that p27 can induce intestinal epithelial cell differentiation has also potentially important implications for cancer in this tissue. Several recent studies in humans have demonstrated abnormally low levels of p27 protein in human carcinomas, correlating well with both histological aggressiveness and patient mortality. Tumors with low or absent p27 have been observed at different sites (breast, prostate, stomach), but most frequently in the intestine (35, 36, 64, 69). Carcinomas with low or absent p27 protein were also found to display enhanced proteolytic activity specific for p27, indicating that more aggressive tumors could have resulted from clonal selection of cells with increased proteasome-mediated degradation rather than altered gene suppression. Only rare instances of homozygous inactivating mutations of the p27 gene have been found in human tumors, but it was recently demonstrated that the murine p27 gene is haploinsufficient for tumor suppression, and both p27 nullizygous and heterozygous mice are predisposed to tumors when challenged with gamma-irradiation or chemical carcinogens (18). The predisposition to intestinal tumors on epigenetic reduction in p27 expression suggests that further elucidation of the mechanism(s) of action of this CKI in intestinal cell differentiation may also reveal new molecular links with the process of intestinal carcinogenesis.


    ACKNOWLEDGEMENTS

We thank Dr. J. F. Beaulieu (University of Sherbrooke, Sherbrooke, Quebec, Canada) for supplying the HIEC6 cells.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-48331.

Address for reprint requests and other correspondence: A. Quaroni, T8 008A VRT, Section of Physiology, Cornell Univ., Ithaca, NY 14853 (E-Mail: aq10{at}cornell.edu).

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

Received 12 January 2000; accepted in final form 26 April 2000.


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