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Am J Physiol Gastrointest Liver Physiol 293: G45-G53, 2007; doi:10.1152/ajpgi.00583.2006
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MUCOSAL BIOLOGY

Downregulation of p63 upon exposure to bile salts and acid in normal and cancer esophageal cells in culture

Sabine Roman,1,2,3,4 Aurélia Pétré,1 Amélie Thépot,1 Agnès Hautefeuille,1 Jean-Yves Scoazec,3,4 François Mion,2,3,4 and Pierre Hainaut1

1Group of Molecular Carcinogenesis and Biomarkers, International Agency for Research on Cancer, Lyon; 2Hospices de Lyon, Digestive Physiology Department, Lyon; 3Claude Bernard University, Lyon I; and 4Institut National de la Santé et de la Recherche Médicale Unité 45, Lyon, France

Submitted 21 December 2006 ; accepted in final form 14 March 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
p63 is a member of the p53 protein family that regulates differentiation and morphogenesis in epithelial tissues and is required for the formation of squamous epithelia. Barrett's mucosa is a glandular metaplasia of the squamous epithelium that develops in the lower esophagus in the context of chronic, gastroesophageal reflux and is considered as a precursor for adenocarcinoma. Normal or squamous cancer esophageal cells were exposed to deoxycholic acid (DCA, 50, 100, or 200 µM) and chenodeoxycholic and taurochenodeoxycholic acid at pH 5. p63 and cyclooxygenase-2 (COX-2) expressions were studied by Western blot and RT-PCR. DCA exposure at pH 5 led to a spectacular decrease in the levels of all isoforms of the p63 proteins. This decrease was observed within minutes of exposure, with a synergistic effect between DCA and acid. Within the same time frame, levels of p63 mRNA were relatively unaffected, whereas levels of COX-2, a marker of stress responses often induced in Barrett's mucosa, were increased. Similar results were obtained with chenodeoxycholic acid but not its taurine conjugate at pH 5. Proteasome inhibition by lactacystin or MG-132 partially blocked the decrease in p63, suggesting a posttranslational degradation mechanism. These results show that combined exposure to bile salt and acid downregulates a critical regulator of squamous differentiation, providing a mechanism to explain the replacement of squamous epithelium by a glandular metaplasia upon exposure of the lower esophagus to gastric reflux.

Barrett's esophagus

BARRETT'S MUCOSA is defined as the replacement of the stratified esophageal squamous epithelium by a metaplastic epithelium containing goblet cells (8). This complication of gastroesophageal reflux disease (GERD) is a premalignant condition that predisposes to esophageal adenocarcinoma, a tumor with an increasing frequency in many Western countries (15). Patients with Barrett's esophagus have an increased bile salt and acid exposure in the lower esophagus in comparison with controls and patients with uncomplicated GERD (26). It has been suggested that bile salt and acid could perturb the differentiation of squamous esophageal cells and promote their reorientation to the glandular differentiation pathway (12). However, the molecular mechanisms responsible for this effect are not known. Understanding these mechanisms and their regulation is essential for the development of successful chemoprevention strategies for the formation of Barrett's mucosa and its transformation into adenocarcinoma.

Barrett's esophagus is a specialized intestinal mucosa containing goblet cells, endoscopically detectable as a salmon pink mucosal segment of irregular shape, contiguous to the esophagogastric junction (11). This mucosa is characterized by a pattern of cytokeratin (CK) expression that combines CK20, a common marker of intestinal differentiation, and CK7, which is considered as a marker of ductal differentiation. In addition, expression of CK13, a marker of squamous mucosa, is lost in Barrett's esophagus (21). Barrett's esophagus has high proliferative indexes associated with altered expression of multiple growth factors, of inducible nitric oxide synthase, and of cyclooxygenase-2 (COX-2; see Ref. 17). The mechanisms of Barrett's esophagus formation are still a matter of conjecture. It has been proposed that, under exposure to gastric reflux, squamous mucosa or associated glandular esophageal ducts may undergo altered differentiation, producing both microvilli and intercellular ridges, resulting in a unique glandular phenotype distinct from adjacent mucosal gastric cells (12). However, the mechanistic basis of the reprogramming of squamous and/or duct cells into cells with glandular features remains to be identified.

The TP63 gene encodes six different protein isoforms with homology to the tumor suppressor protein p53. The strongest homology lies in the DNA-binding domain, which is common to all isoforms (4). The isoforms differ in their NH2-terminal and/or COOH-terminal domains. In the NH2 terminus, two types of isoforms can be distinguished, transcriptionally active (TA), which contains the NH2-terminal main transcriptional activation domain, and {Delta}N, which lacks the TA domain because of usage of an alternative promoter located in intron 3. In the COOH terminus, alternative splicing generates three distinct variants of different length ({alpha}, beta, and {gamma}; see Ref. 1). Although the exact role of each isoforms is ill-defined, there is evidence that spatial and temporal compartmentalization of the expression of TA and {Delta}N isoforms is essential for keratinocyte stem cell differentiation and proliferation (1). Specifically, {Delta}N p63 isoforms are strongly expressed in proliferative stem cells and keratinocytes, whereas expression of TA isoforms occurs in suprabasal cells during the commitment to terminal differentiation (6). Mice lacking TP63 (p63–/– mice) show a number of defects in epithelial and mesenchymal differentiation and morphogenesis. In particular, they lack normal skin development because of the inability of skin keratinocytes to establish a stable, stratified epithelium. Of note, the esophageal lining of these mice shows a pseudostratified columnar appearance with abundance of goblet cells (28), similar to Barrett's esophagus. This observation, in conjunction with the fact that p63 is expressed in the basal and suprabasal layers of human esophageal squamous epithelium but not in Barrett's esophagus (10), leads us to suggest that downregulation of p63 expression may represent a critical molecular event in Barrett's esophagus formation.

In this study, we have exposed squamous esophageal cells, either primary cultures or squamous cell carcinoma cell lines, to treatments with bile salts and acid that mimic the effects of gastric reflux. We show that these treatments induce the rapid downregulation of p63 proteins through a mechanism partially blocked by proteasome inhibitors. These results show that components of gastric refluxes activate a pathway that destabilizes p63, providing a plausible mechanism for the reprogramming of squamous esophageal stem cells into intestinal-type cells during the early steps of Barrett's esophagus formation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell cultures and treatments. The esophageal squamous carcinoma cell lines TE-1 and TE-13 (3, 19) were used. TE-cells were grown in RPMI 1640 medium (GIBCO) supplemented with 10% heated FBS, 1% antibiotics (penicillin, streptomycin), and 2 nM L-glutamine and were maintained as a subconfluent monolayer in a humidified incubator at 37°C (or 32°C for TE-1) in the presence of 10% CO2. Barrett's associated adenocarcinoma cells lines, Bic-1 (mutant p53) and Seg-1 (wild-type p53), were grown in DMEM (GIBCO) supplemented with 10% heated FBS, 1% antibiotics (penicillin, streptomycin), and 2 nM L-glutamine in a humidified incubator at 37°C in the presence of 5% CO2. Primary esophageal epithelial cells were isolated from segments of normal esophagus obtained during multiorgan harvesting for transplantation under conditions approved by an ethics committee according to French Law. These cells were grown on feeder layers of human skin fibroblasts in DMEM with glutamax (GIBCO) and Ham's F-12 (GIBCO) supplemented with FBS (10%), penicillin (100 IU/ml), gentamicin (20 µg/ml), amphotericin B (1 µg/ml), epithelial growth factor (10 ng/ml), hydrocortisone (0.4 µg/ml), insulin (0.12 IU/ml), isoprenaline (0.4 µg/ml), triiodothyronine (2.10–9 M), and adenine (24.3 µg/ml) in a humidified incubator at 37°C in the presence of 5% CO2.

To mimic the effect of bile salts and acid reflux, TE cells were exposed to bile acids and acid. Gastroesophageal reflux juice contains both conjugated and unconjugated bile acids. Bile salts were selected based on the composition of esophageal aspirates of patients with Barrett's esophagus, who have been shown to have a significantly greater proportion of deoxycholic acid (DCA) and taurine-conjugated forms in their refluxate (18). The bile salts used were DCA (50, 100 or 200 µM), chenodeoxycholic acid (CDCA, 200 µM), or taurochenodeoxycholic acid (TCDCA, 200 µM) (Sigma, St. Louis, MO) diluted in serum-free medium. For treatment with acid, the culture medium was acidified with 1 N HCl, a pH lower than the pKa for DCA, which is pH 6. Bile salts and/or acid were added in the culture medium for 24 h. To better reflect the physiopathological duration of reflux episodes in patients, cells were exposed to bile salts and/or acid for pulses of 10 min to 1 h, followed by medium replacement and 23 h of further culture in serum-free medium. Time-course experiments with bile salt and acid exposures ranging from 5 to 80 min were also performed, and cells were harvested immediately after. For genotoxic treatments, cells were exposed for 24 h with different doses of doxorubicin (250, 500, and 1,000 ng/ml) added to the culture medium.

Lactacystin (20 µM; Sigma) or 50 µM MG-132 (Calbiochem) were added to the culture medium 6 h before cell exposure to DCA and acid exposure to inhibit proteasome activity. Measurements of cyclin A levels were used as a control of the efficiency of proteasome inhibition.

Flow cytometry. Cell cycle was analyzed by flow cytometry using FACsCalibur (Becton-Dickinson, Mountain View, CA) and a Cycle TEST PLUS staining kit (Becton-Dickinson) according to the manufacturer's instructions. Cell cycle analysis was repeated in four separate experiments. The percentage of sub-G1 cells, indicative of apoptosis, was expressed as means (+SD) and compared by paired t-test with the percentage of apoptotic cells before DCA (50 µM, pH 5) exposure. A difference was considered as significant if P < 0.05.

Trypan blue assay. TE cells were cultured in six-well plates (400,000 cells/well). After 5 to 80 min exposure to 50 µM DCA at pH 5, cells were harvested with trypsin and counted after trypan blue staining. The results are expressed as a percent increase in the number of dead cells over control. A mean of nine experiments per condition was calculated and compared with control using paired t-test. A difference was considered as significant if P < 0.05.

Clonogenic assay. TE-1 37°C were exposed 5–80 min to 50 µM DCA at pH 5. At the end of exposure, cells were harvested by trypsinization, and 2,000 cells were cultured in 60-mm plates in RPMI 1640 medium (GIBCO) supplemented with 10% heated FBS, 1% antibiotics (penicillin, streptomycin), and 2 nM L-glutamine. Medium was changed every 3 days. After 10 days, plates were stained with Giemsa.

Total protein extraction and Western blot analysis. Cells were lysed by adding lysis buffer [50 mM Tris·HCl, pH 7.4, 250 mM NaCl, 0.1% SDS, 0.5% Nonidet P-40 (NP-40), and protease inhibitors (2 µg/ml aprotinin, 500 µM phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, 1 pg/ml pepstatin and 2 mM dithiothreitol)] and placed on ice for 30 min. Extracts were subsequently centrifuged at 13,000 rpm for 15 min at 4°C. Supernatants were then collected. Protein concentration was determined using the method reported by Bradford (5).

Whole protein extracts were loaded, separated by SDS-PAGE with the use of a 7.5%, gel and transferred to polyvinylidine difluoride membranes by electroblotting. Membranes were blocked with 5% dry milk in PBS-NP-40 (0.05%) for 1 h at room temperature and incubated overnight with primary antibody diluted in PBS-NP-40 containing 1% dry milk. The following antibodies were used: mouse monoclonal anti-p63 at 1:500 (4A4, Ab1; Oncogene), which recognizes all p63 isoforms; rabbit polyclonal anti-TAp63 at 1:500 (Biolegend), which recognizes TAp63 isoforms; rabbit polyclonal anti-{Delta}Np63 at 1:1,000 (anti-P40; Calbiochem), which recognizes {Delta}Np63 isoforms; rabbit polyclonal anti-{alpha}-p63 at 1:500 (H129; Santa Cruz), which recognizes p63 {alpha}-isoforms; mouse monoclonal anti-p53 at 1:3,000 (DO7; Dako); goat polyclonal anti-COX 2 at 1:1,000 (C-20; Santa Cruz); mouse monoclonal anti-CK13 at 1:500 (Chemicon); mouse monoclonal anti-CK13 at 1:500 (Chemicon); rabbit polyclonal anti-cyclin A at 1:1,000 (H-432; Santa Cruz); mouse monoclonal anti-actin at 1:20,000 (C-4; ICN Biomedicals); or rabbit polyclonal anti-Ku80 at 1:40,000 (Serotec). The membranes were washed and incubated with peroxidase-conjugated secondary antibody, either goat anti-mouse at 1:10,000 (Dako), donkey anti-goat at 1:5,000 (Santa Cruz), or goat anti-rabbit at 1:3,000 (Dako). Proteins were detected by chemiluminescence (Amersham Biosciences). Actin and Ku80 were used as protein loading controls.

RNA extraction and RT-PCR. Total mRNA from cells was extracted by using Tri-Zol Reagent (Invitrogen), and first-strand cDNA was synthesized from total RNA using random hexamer primers and the SuperScript II system for RT-PCR (Invitrogen). TAP63 cDNA (forward 5'-GACCTGAGTgACCCCATGTG-3' and reverse 5'-CGGGTGATGGAGAGAGAGCA-3') and {Delta}NP63 cDNA (forward 5'-TGCCCAGACTCAA TTTAGTGAG-3' and reverse 5'-AGAGAGAGCATCGAAGGTGGAG-3') were coamplified with beta-actin (forward 5'-GACACCACTGGAGGGTGACT-3' and reverse 5'-CCACATGGTT CTTCCTCTGCT-3') for semiquantitative analysis. PCR was performed for 25 cycles for beta-actin, for 28 cycles for {Delta}Np63, and for 30 cycles for TAp63. Amplified products were analyzed on 2% agarose gels. Real-time PCR was also performed with ABI Prism 7900 HT Sequence Detection System (Applied Biosystems, Courtaboeuf, France). Relative quantification was performed in a multiplex reaction using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a housekeeping, control gene (24). The primers and fluorogenic probes for PCR reaction were the following: TAp63 forward 5'-CACACAGACAAAT GAATTCCTCAGT-3', reverse 5'-CATCCACAAAGTTCAAGTCAA TGGG-3'/5' 6-FAM-GGGATTTTCTGGAACAGC-MGB and {Delta}Np63 forward 5'-GGAAA ACAATGCCCAGACTCA-3', reverse 5'-TGTTCAGGAGCCCCAGGTT-3'/5' 6-FAM-TTA GTGAGCCACA GTACAC-MGB. Each sample was analyzed in duplicate, and each experiment was done in triplicate. Relative quantification was performed using the comparative threshold cycle (CT) method as described in the User Bulletin, ABI Prism 7900 HT Sequence Detection System. The relative expression level of the TP63 gene in the different conditions was estimated by calculating the {delta}CT value, defined as the difference in the CT value for the target (TAP63 or {Delta}NP63) and the reference gene (GAPDH; see Ref. 9). Experiments were done in triplicate. Results were expressed as the mean of {delta}CT (+SD) and compared with paired t-test. A difference was considered as significant if P < 0.05.

Detection of Intracellular Localization of p63 by Immunofluorescence Immunofluorescence was performed on cells grown on glass slides and fixed in 4% formaldehyde for 30 min at room temperature. After washing with PBS, cells were permeabilized with 1% Triton X-100 for 10 min and then washed with PBS. After being blocked with 1% BSA in PBS for 1 h, cells were washed with PBS and incubated with rabbit polyclonal anti-p63 antibody (H137; which recognizes all p63 isoforms; Santa Cruz) at room temperature for 1.5 h. After being washed with PBS three times, cells were incubated with a secondary antibody (FITC-labeled anti-rabbit immunoglobulins) at room temperature for 1 h. After being washed with PBS three times, cells were incubated with 4',6-diamino-2-phenylindole (DAPI, 10 mg/ml; Sigma-Aldrich) for 3 min and washed again with PBS. Finally, the slides were mounted, and cells were examined under a fluorescent microscope.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of p63 isoforms expressed in TE cells. The esophageal squamous carcinoma cell lines TE-1 and TE-13 (19) both express various p63 isoforms. Figure 1 shows the profiles of p63 isoform expressions using different antibodies. As a reference to identifying isoforms, cloned cDNA corresponding to each isoform were transfected in Hep 3B, a hepatocellular carcinoma cell line that expresses low levels of p63, and analyzed by Western Blot. The TE-1 expresses a temperature-sensitive mutant p53, V272M, that adopts a wild-type conformation at 32°C and a mutant conformation with loss of DNA binding at 37°C (16). We have used this cell line to assess the effect of exposure to acid and bile in cells expressing either active (wild-type) or inactive (mutant) p53 protein. The temperature shift does not significantly alter the expression of p63 (Fig. 1). TE-13 lacks p53 protein expression because of homozygous mutation at the donor splice site of exon 5 and abnormal splicing (3). In both cell lines, the most abundant p63 isoform is {Delta}Np63{alpha} (70 kDa; Fig. 1). Faint bands may correspond to {Delta}Np63beta and TAp63{gamma} isoforms.


Figure 1
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  		Fig. 1. Profiles of p63 isoform expression. Lanes 1–6: cloned cDNA corresponding to different p63 isoforms transfected in Hep 3B and analyzed by Western Blot; lane 7: TE-13 cells; lanes 8 and 9: TE-1 cells at 37 and 32°C, respectively. Different antibodies against p63 were used as follows: 4A4, which recognizes all p63 isoforms; anti-TAp63, which recognizes transcriptionally active (TA) isoforms; anti-{Delta}Np63 which recognizes {Delta}N isoforms; and anti-p63{alpha}, which recognizes p63 {alpha}-isoforms. Arrows on right indicate the expected position of the major p63 isoforms. {Delta}N{alpha} isoform appears to be the predominant p63 isoform in TE-1 at 32°C, at 37°C, and in TE-13.

 
Synergistic effect of bile salts and acid exposure on p63 and p53 expression in TE cells and in primary epithelial esophageal cells. First, we have exposed esophageal squamous cells to bile salts and acid, either separately or together, and we have examined their effects on the expression of p63. In TE-13 cells, p63 decreased drastically after 24 h exposure to DCA and acid, whereas acid alone had no effect and DCA alone only slightly decreased p63 expression (Fig. 2A). Similar results were observed in TE-1 cells at 32°C and 37°C after a 1-h pulse of DCA and/or acid (Fig. 2B). In these cells, a p53 decrease in p53 levels was also observed upon DCA and acid exposure. Next, we compared the changes in p63 levels with those of COX-2, an enzyme typically induced by reflux stress and expressed at high levels in Barrett's esophagus (13). The decrease in p63 levels was associated with an increase in COX-2 protein levels in TE-cells exposed to a 1-h pulse of DCA. DCA increased COX-2 expression both at pH 5 and pH 7. COX-2 expression was also slightly increased at pH 5 alone. The same results were observed in TE-13 exposed for 24 h continuously or for a 10-min pulse, as well as after a 1-h pulse in TE-13 and in TE-1 at both 32°C and 37°C (Fig. 2, A and B).


Figure 2
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  		Fig. 2. Western Blot analysis of synergistic effect of deoxycholic acid (DCA) and acid exposure on p63, p53, and cyclooxygenase-2 (COX-2) expression in TE cells. A: TE-13 were exposed to bile salts (100 or 200 µM DCA) at pH 7 or 5 during 10 min followed by 23 h serum-free medium, or for 24 h. B: TE-1 at 32°C and 37°C were exposed for 1 h to bile salts (100 or 200 µM DCA) at pH 7 or 5 followed by 23 h serum-free medium. p63 protein expression was studied using 4A4, an antibody that recognizes all p63 isoforms. Actin detection was used as a loading control.

 
A decrease in p63 levels was also observed in monolayer cultures of primary esophageal epithelial cells (Fig. 3). In contrast with squamous cells, treatments with acid and bile increased p53 protein levels in Bic-1 and had only small effects at higher doses in Seg-1, two cell lines derived from Barrett's adenocarcinoma (Fig. 3). The former expresses a mutant p53 protein, whereas the latter expresses wild-type p53, explaining the variations in p53 levels between the two lines. These two cell lines did not express detectable levels of p63 proteins.


Figure 3
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  		Fig. 3. Effects of bile salts and acid in primary esophageal cells and in adenocarcinoma cell lines. Primary esophageal epithelial cells and esophageal adenocarcinoma cell lines, Bic-1 and Seg-1, were exposed for 1 h to DCA at pH 5 or 7. p53 and p63 protein levels were analyzed by Western blot using DO7, an antibody that recognizes p53, and 4A4, which recognizes all p63 isoforms. Actin or Ku80 were used as control of protein loading.

 
To assess whether the decrease in p63 protein levels was associated with a decrease in other markers of squamous cell differentiation, we analyzed the expression of CK13 after exposure of TE-1 cells at 32°C or 37°C to DCA and acid for 1 h. CK13 is expressed in squamous mucosa but not in glandular mucosa. Figure 4 shows that this treatment induces a small but consistent decrease in the levels of CK13, suggesting that the rapid decrease in p63 levels does not result from a general downregulation of all markers of squamous differentiation.


Figure 4
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  		Fig. 4. Western blot analysis of cytokeratin 13 (CK13) expression in TE-1 cells after 1 h pulse exposure to DCA and acid. TE-1 cells were exposed for 1 h to DCA at pH 5 or 7 followed by 23 h serum-free medium. CK13 was detected using a mouse monoclonal anti-CK13 antibody. Actin or Ku80 were used as control of protein loading.

 
Compatible results were obtained with CDCA. However, exposure to the taurine conjugate, TCDCA, did not induce a decrease in p63 levels in TE cells (Fig. 5). Together, these results indicate that combined treatment with unconjugated bile salts and acid induces a spectacular downregulation of p63 protein, occurring in the same time frame as the increase in COX-2 expression, which is characteristic of esophageal squamous cells exposed to reflux stress.


Figure 5
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  		Fig. 5. p63 expression in response to different bile salts at pH 5. TE cells were exposed for 5–80 min to an unconjugated primary bile acid [200 µM chenodeoxycholic acid (CDCA)] or to a conjugated primary bile acid [200 µM taurochenodeoxycholic acid (TCDCA)]. p63 protein expression was analyzed by Western blot using 4A4, an antibody that recognizes all p63 isoforms. Actin was used as a control of protein loading.

 
Mechanisms of DCA- and acid-induced p63 downregulation. To address the mechanism of the observed decrease in p63 levels, we first sought to determine whether this decrease was occurring in a context of general cytotoxicity. In Fig. 6, we show that p63 and p53 protein levels significantly decreased after 10 or 20 min of exposure to DCA and acid. Flow cytometry analysis within this time frame did not reveal substantial changes in cell-cycle distribution nor an increase in sub-G1 apoptotic cells, as shown in TE-1 at 37°C (Fig. 7, A and B). Using trypan blue exclusion, cell viability was found to be preserved for up to 80 min of treatment, despite a small increase in the percentage of dead cells (Fig. 7C). Finally, Western blot analysis did not reveal an increase in the cleavage of poly-ADP-ribose-polymerase, a substrate of caspase 3 that is an early marker of apoptosis (data not shown). Thus the decrease in p63 observed within 10–20 min of exposure to acid and bile salts cannot be ascribed to generalized cell death. Further assessment of cell survival using a clonogenic assay was carried out after treatment of cells with bile salts and acids for up to 80 min, followed by long-term culture in fresh medium. These experiments showed that cells kept the ability to clonally proliferate even after 80 min of exposure (data not shown).


Figure 6
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  		Fig. 6. Time course of p53 and p63 decrease. TE cells were exposed for 5, 10, 20, 40, or 80 min to 50 µM DCA at pH 5. p63 and p53 were detected by Western blotting using 4A4 and DO7, respectively. Actin was used as control of protein loading.

 

Figure 7
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  		Fig. 7. DCA and acid induce a moderate cytotoxicity in TE cells. A: cell cycle was analyzed by flow cytometry in TE-1 cells at 37°C after labeling with propidium iodide. No difference in cell cycle distribution was observed before and after 20 min exposure to 50 µM DCA at pH 5. B: percentage of apoptotic cells was estimated using the percentage of sub-G1 cells detected by flow cytometry (mean + SD) in TE-1 cells exposed for 5–80 min to 50 µM DCA at pH 5. The percentage of sub-G1 cells did not differ significantly before and after treatment (paired t-test, P > 0.05). C: cell viability was assessed in TE cells after 5–80 min of exposure to DCA and acid using trypan blue exclusion. The results were expressed as a percent increase in the number of dead cells over control. A mean of 9 experiments/condition was calculated and compared with control using paired t-test. The percentage of dead cells is significantly higher in all conditions in comparison with untreated cells (P < 0.05) but did not differ with time of DCA and acid exposure (5–80 min).

 
To determine whether the reduction in p63 levels was a common response to genotoxic stress damage, we analyzed the effects of treatment with doxorubicin, an inhibitor of topoisomerase II that induces single-strand DNA breaks. Figure 8 shows that this treatment did not induce the same extent of p63 downregulation as bile salts and acid. However, a small, dose-dependent decrease in p63 was detected, resulting in a small reduction of the p63 signal detected by Western blot after treatment with doxorubicin at 1 µg/ml.


Figure 8
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  		Fig. 8. Effect of doxorubicine on p63, p53, and COX-2 protein level in TE cells. p53, p63, and COX-2 expression was studied by Western blot after 24 h exposure to different doses of doxorubicin (250, 500, and 1,000 ng/ml). The following antibodies were used: 4A4, which recognizes all p63 isoforms; DO7, which recognizes p53; and C-20, which recognizes COX-2. Actin was used as a control of protein loading.

 
Next, we analyzed p63 mRNA levels within minutes of exposure to determine whether the decrease in protein levels was preceded by a similar decrease in mRNA levels. We found that p63 mRNA levels were not significantly reduced by DCA and acid exposure (Fig. 9A). Real-time, quantitative PCR showed that DCA and acid exposure did not significantly modify the respective levels of TA and {Delta}Np63 transcripts (Fig. 9B). Thus the decrease in p63 protein levels does not appear to result from a shut down of transcription.


Figure 9
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  		Fig. 9. TA and {Delta}Np63 transcripts after DCA exposure. A: qualitative PCR of p63 mRNA in TE-1 cells at 37°C after 5, 10, and 20 min exposure to 50 µM DCA at pH 5. The expression of TA and {Delta}Np63 isoforms is shown on RT-PCR gel. Actin is used as a PCR control. B: real-time PCR in TE cells exposed for 20 min to 50 µM DCA at pH 5. Relative expression levels of TA and {Delta}Np63 in comparison with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), expressed as {Delta}threshold cycle (CT) values, are not significantly modified after 20 min exposure of DCA at pH 5 (paired t-test, P > 0.05).

 
To determine whether the apparent drop in p63 levels may be because of the relocalization of the protein in a different cell compartment, the localization of p63 after DCA and acid exposure was analyzed by immunofluorescence. At baseline, p63 was expressed in the nucleus of the cells. After 20 min of DCA and acid exposure, p63 staining was clearly decreased in the nucleus and slightly increased in the cytoplasm, indicating that the protein may undergo a drift from nucleus to cytoplasm (Fig. 10). Finally, we used proteasome inhibitors to determine whether the decrease in p63 protein may be the result of active protein degradation by the proteasome. We found that MG-132 prevented the decrease in p53 and p63 levels induced by DCA and acid (Fig. 11). Similar results were observed when proteasome was inhibited by lactacystin (data not shown).


Figure 10
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  		Fig. 10. p63 immunofluorescence in TE-13 cells. In the absence of treatment, p63 immunofluorescence was expressed in the nucleus, whereas it was detectable in the cytoplasm after 20 min of exposure to DCA at pH 5. 4',6-Diamino-2-phenylindole (DAPI) was used to stain nuclei.

 

Figure 11
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  		Fig. 11. Proteasome-dependent degradation of p63 and p53 after treatment with bile salts and acid in TE-1 cells at 32°C. Western blot analysis of p63 (antibody 4A4) or p53 (antibody DO7) after exposure to DCA at pH 5 (0–40 min) in the presence (+) or absence (–) of MG-132 (50 µM), a proteasome inhibitor. Cyclin A was used as a positive control of proteasomal inhibition efficiency. Actin was used as loading control.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The mechanisms that lead to the formation of Barrett's esophagus imply the reprogramming of differentiation patterns of the esophageal mucosa under stress induced by reflux. This process is likely to involve at least two steps, with first the reorientation of differentiation from squamous to glandular patterns and second the selection and survival of the latter cell type as the fittest to survive in a reflux environment. In recent years, compelling evidence has accumulated that the p63 protein is an essential regulator of squamous differentiation. In the present study, we have tested whether in vitro treatments mimicking reflux conditions may affect the status of p63 in esophageal squamous cells. Our results clearly show that bile and acid exposure can downregulate both p53 and p63 protein levels in esophageal cell lines as well as in primary esophageal cell cultures, in conditions in which a characteristic marker of stress and inflammatory responses, COX-2, is induced. This observation suggests that downregulation of p63 may be a critical adaptive response of squamous cells to stress by reflux.

The p63 protein and its various isoforms play a critical role in the development and maintenance of stratified epithelial tissues (6). Using a loss of function approach, Barbieri and colleagues (2) showed that disruption of p63 expression in squamous cell lines and keratinocytes led to downregulation of markers of squamous epithelium and that keratinocytes with reduced p63 expression failed to properly differentiate in vitro. This observation is compatible with the notion that p63 is indeed required for squamous differentiation. In agreement with this notion, we have observed that the decrease in p63 induced by treatment with bile salts and acid was followed by a small but consistent reduction in the expression of CK13, a typical marker of squamous differentiation. Thus the rapid downregulation of p63 by bile and acid may represent a very early event in the sequence of events leading from squamous esophageal epithelium to Barrett's metaplasia.

In humans, it has been shown that the toxicity of bile acids toward the esophageal mucosa is dependent on pH (18). In vitro studies have suggested that bile and acid have synergistic effects (25). In concordance with this idea, we have observed a synergistic effect of unconjugated primary or secondary bile acids (CDCA and DCA, respectively) and pH 5 in inducing a decrease in p53 and p63 protein levels. The biological properties of bile salt are strongly pH dependent. Bile acids are partly in an unionized state and are soluble at pH below pKa of bile salt. Only unionized forms of bile acid can penetrate through cell membranes. In contrast, unconjugated bile acids have a pKa around 6 (7). Thus, at pH 5, DCA and CDCA are capable of penetrating through cell membranes and exerting their effects. On the contrary, TCDCA, insoluble at pH 5 (pKa = 2), had no effect on p63 and p53 expression, suggesting that bile salts have to penetrate into cells to modify p63 expression.

DCA and acid exposure induced COX-2 expression in esophageal cell lines. This result is consistent with previous results (14, 22). This observation indicates that the decrease in p63 levels occurs in the same general signaling context as the one that is responsible for the increase of COX-2 protein levels. Whether this result is because of a role of p63 as an inhibitor of COX-2 expression or because of the fact that decreasing p63 levels in squamous cells induces an overall cellular stress situation remains to be investigated.

This study demonstrates that p63 protein isoforms may undergo regulation by proteasome-dependent degradation in response to stress by bile and acid. Regulation of p63 by degradation has also been reported in response to genotoxic agents. Westfall and colleagues (27) have observed a downregulation of {Delta}Np63{alpha} after joint exposure to ultraviolet (UV)-C and paclitaxel of primary keratinocytes or SCC cell lines. Furthermore, they found that this degradation was proteasome dependent and implied the ubiquitination of {Delta}Np63{alpha}. It should be noted that there is evidence that UV-C irradiation also increases the expression of TAp63{alpha} in mouse erythroleukemia cells (20).

Posttranslational modifications, including phosphorylation, ubiquitination, and sumoylation, play critical roles in the regulation of p63 stability and proteasomal degradation (1). For example, the UV-induced decrease of {Delta}Np63{alpha} is accompanied by an increased phosphorylation and ubiquitination of {Delta}Np63{alpha} (27). In the present study, we have observed that treatment with bile and acid in the presence of proteasome inhibitors induced an increase in high-molecular-weight forms of the p63 protein (data not shown). Whether these forms correspond to ubiquitinylated proteins remains to be demonstrated. We have also shown that DCA reduced the levels of nuclear p63 protein. Thus DCA may facilitate nuclear export of p63 into the cytoplasm of squamous cells, similar to the export described for p53 after exposure of colonic cells to DCA, which was correlated with stimulated proteasomal degradation (23). In our study, a decrease in p63 levels was paralleled by a decrease in p53 levels, suggesting that the same mechanisms may be involved in controlling the stability of both proteins in response to stress by bile and acid. Interestingly, this degradation pathway appears to be cell-type specific, since DCA treatment did not induce a similar drop in p53 levels in two esophageal cell lines derived from Barrett's adenocarcinoma, Bic-1 and Seg-1.

In conclusion, we show here that DCA at pH 5 decreased p63 protein levels by inducing its degradation by the proteasome and increased COX-2 protein levels in esophageal cell lines and primary esophageal cell cultures. These observations are consistent with the hypothesis that specific downregulation of p63 by bile and acid may represent a primary event in response to reflux stress. Loss of p63 in squamous cells may prevent adequate cicatrisation of ulcerated areas of the esophageal mucosa, leading to the replacement of squamous by glandular cells, a type of cell that is more resistant to reflux stress. It is also possible that stress-induced degradation of p63 facilitates the differentiation of surviving stem cells into a glandular differentiation pathway.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Roman, Digestive Physiology, Pavillon H, Hopital Edouard Herriot, Place d'Arsonval, 69437 Lyon Cedex 03, France (e-mail: sabine.roman{at}chu-lyon.fr)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Barbieri CE, Pietenpol JA. p63 and epithelial biology. Exp Cell Res 312: 695–706, 2006.[CrossRef][Web of Science][Medline]
  2. Barbieri CE, Tang LJ, Brown KA, Pietenpol JA. Loss of p63 leads to increased cell migration and up-regulation of genes involved in invasion and metastasis. Cancer Res 66: 7589–7597, 2006.[Abstract/Free Full Text]
  3. Barnas C, Martel-Planche G, Furukawa Y, Hollstein M, Montesano R, Hainaut P. Inactivation of the p53 protein in cell lines derived from human esophageal cancers. Int J Cancer 71: 79–87, 1997.[CrossRef][Web of Science][Medline]
  4. Benard J, Douc-Rasy S, Ahomadegbe JC. TP53 family members and human cancers. Hum Mutat 21: 182–191, 2003.[CrossRef][Web of Science][Medline]
  5. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254, 1976.[CrossRef][Web of Science][Medline]
  6. Daniely Y, Liao G, Dixon D, Linnoila RI, Lori A, Randell SH, Oren M, Jetten AM. Critical role of p63 in the development of a normal esophageal and tracheobronchial epithelium. Am J Physiol Cell Physiol 287: C171–C181, 2004.[Abstract/Free Full Text]
  7. DeMeester TR. Clinical biology of the Barrett's metaplasia, dysplasia to carcinoma sequence. Surg Oncol 10: 91–102, 2001.[CrossRef][Web of Science][Medline]
  8. Flejou JF. Barrett's oesophagus: from metaplasia to dysplasia and cancer. Gut 54, Suppl 1: i6–12, 2005.[Abstract/Free Full Text]
  9. Gemignani F, Moreno V, Landi S, Moullan N, Chabrier A, Gutierrez-Enriquez S, Hall J, Guino E, Peinado MA, Capella G, Canzian F. A TP53 polymorphism is associated with increased risk of colorectal cancer and with reduced levels of TP53 mRNA. Oncogene 23: 1954–1956, 2004.[CrossRef][Web of Science][Medline]
  10. Glickman JN, Yang A, Shahsafaei A, McKeon F, Odze RD. Expression of p53-related protein p63 in the gastrointestinal tract and in esophageal metaplastic and neoplastic disorders. Hum Pathol 32: 1157–1165, 2001.[CrossRef][Web of Science][Medline]
  11. Haag S, Holtmann G. Reflux disease and Barrett's esophagus. Endoscopy 35: 112–117, 2003.[Medline]
  12. Jankowski JA, Harrison RF, Perry I, Balkwill F, Tselepis C. Barrett's metaplasia. Lancet 356: 2079–2085, 2000.[CrossRef][Web of Science][Medline]
  13. Jenkins GJ, Doak SH, Parry JM, D'Souza FR, Griffiths AP, Baxter JN. Genetic pathways involved in the progression of Barrett's metaplasia to adenocarcinoma. Br J Surg 89: 824–837, 2002.[CrossRef][Web of Science][Medline]
  14. Kaur BS, Ouatu-Lascar R, Omary MB, Triadafilopoulos G. Bile salts induce or blunt cell proliferation in Barrett's esophagus in an acid-dependent fashion. Am J Physiol Gastrointest Liver Physiol 278: G1000–G1009, 2000.[Abstract/Free Full Text]
  15. Lagergren J. Adenocarcinoma of oesophagus: what exactly is the size of the problem and who is at risk? Gut 54, Suppl 1: i1–5, 2005.[Abstract/Free Full Text]
  16. Maurici D, Monti P, Campomenosi P, North S, Frebourg T, Fronza G, Hainaut P. Amifostine (WR2721) restores transcriptional activity of specific p53 mutant proteins in a yeast functional assay. Oncogene 20: 3533–3540, 2001.[CrossRef][Web of Science][Medline]
  17. Morales CP, Souza RF, Spechler SJ. Hallmarks of cancer progression in Barrett's oesophagus. Lancet 360: 1587–1589, 2002.[CrossRef][Web of Science][Medline]
  18. Nehra D, Howell P, Williams CP, Pye JK, Beynon J. Toxic bile acids in gastro-oesophageal reflux disease: influence of gastric acidity. Gut 44: 598–602, 1999.[Abstract/Free Full Text]
  19. Nishihira T, Hashimoto Y, Katayama M, Mori S, Kuroki T. Molecular and cellular features of esophageal cancer cells. J Cancer Res Clin Oncol 119: 441–449, 1993.[CrossRef][Web of Science][Medline]
  20. Okada Y, Osada M, Kurata S, Sato S, Aisaki K, Kageyama Y, Kihara K, Ikawa Y, Katoh I. p53 gene family p51(p63)-encoded, secondary transactivator p51B(TAp63alpha) occurs without forming an immunoprecipitable complex with MDM2, but responds to genotoxic stress by accumulation. Exp Cell Res 276: 194–200, 2002.[CrossRef][Web of Science][Medline]
  21. Ormsby AH, Goldblum JR, Rice TW, Richter JE, Falk GW, Vaezi MF, Gramlich TL. Cytokeratin subsets can reliably distinguish Barrett's esophagus from intestinal metaplasia of the stomach. Hum Pathol 30: 288–294, 1999.[CrossRef][Web of Science][Medline]
  22. Ouatu-Lascar R, Fitzgerald RC, Triadafilopoulos G. Differentiation and proliferation in Barrett's esophagus and the effects of acid suppression. Gastroenterology 117: 327–335, 1999.[CrossRef][Web of Science][Medline]
  23. Qiao D, Gaitonde SV, Qi W, Martinez JD. Deoxycholic acid suppresses p53 by stimulating proteasome-mediated p53 protein degradation. Carcinogenesis 22: 957–964, 2001.[Abstract/Free Full Text]
  24. Rubie C, Kempf K, Hans J, Su T, Tilton B, Georg T, Brittner B, Ludwig B, Schilling M. Housekeeping gene variability in normal and cancerous colorectal, pancreatic, esophageal, gastric and hepatic tissues. Mol Cell Probes 19: 101–109, 2005.[CrossRef][Web of Science][Medline]
  25. Tselepis C, Morris CD, Wakelin D, Hardy R, Perry I, Luong QT, Harper E, Harrison R, Attwood SE, Jankowski JA. Upregulation of the oncogene c-myc in Barrett's adenocarcinoma: induction of c-myc by acidified bile acid in vitro. Gut 52: 174–180, 2003.[Abstract/Free Full Text]
  26. Vaezi MF, Richter JE. Role of acid and duodenogastroesophageal reflux in gastroesophageal reflux disease. Gastroenterology 111: 1192–1199, 1996.[CrossRef][Web of Science][Medline]
  27. Westfall MD, Joyner AS, Barbieri CE, Livingstone M, Pietenpol JA. Ultraviolet radiation induces phosphorylation and ubiquitin-mediated degradation of DeltaNp63alpha. Cell Cycle 4: 710–716, 2005.[Web of Science][Medline]
  28. Yang A, Schweitzer R, Sun D, Kaghad M, Walker N, Bronson RT, Tabin C, Sharpe A, Caput D, Crum C, McKeon F. p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 398: 714–718, 1999.[CrossRef][Medline]




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