A novel pathological role of p53 in kidney development revealed by gene-environment interactions
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《美国生理学杂志》
Department of Pediatrics, Section of Pediatric Nephrology, Tulane University Health Sciences Center, New Orleans, Louisiana
ABSTRACT
Gene-environment interactions are implicated in congenital human disorders. Accordingly, there is a pressing need to develop animal models of human disease, which are the product of defined gene-environment interactions. Previously, our laboratory demonstrated that gestational salt stress of bradykinin B2 receptor (B2R)-null mice induces renal dysgenesis and early death of the offspring (El-Dahr SS, Harrison-Bernard LM, Dipp S, Yosipiv IV, and Meleg-Smith S. Physiol Genomics 3: 121–131, 2000). In contrast, salt-stressed B2R +/+ or +/– littermates have normal development. The present study investigates the mechanisms underlying the susceptibility of B2R-null mice to renal dysgenesis. Proteomic and conventional Western blot screens identified E-cadherin among the differentially repressed proteins in B2R–/– kidneys, whereas the checkpoint kinase Chk1 and its substrate P-Ser20 p53 were induced. We tested the hypothesis that p53 mediates repression of E-cadherin gene expression and is causally linked to the renal dysgenesis. Genetic crosses between B2R –/– and p53+/– mice revealed that germline reduction of p53 gene dosage rescues B2R–/– mice from renal dysgenesis and restores kidney E-cadherin gene expression. Furthermore, -irradiation induces repression of E-cadherin gene expression in p53+/+ but not –/– cells. In transient transfection assays, p53 repressed human E-cadherin promoter-driven reporter activity, whereas a mutant p53, which cannot bind DNA, did not. Functional promoter analysis indicated the presence of a p53-responsive element in exon 1, which partially mediates p53-induced repression. Chromatin immunoprecipitation assays revealed that p53 inhibits histone acetylation of the E-cadherin promoter. Treatment with a histone deacetylase inhibitor reversed both p53-mediated promoter repression and deacetylation. In conclusion, this study demonstrates that gene-environment interactions cooperate to induce congenital defects through p53 activation.
bradykinin B2 receptor; knockout mice; checkpoint kinase; histone acetylation
CONGENITAL RENAL DYSGENESIS, leading to hypoplasia, dysplasia, and cystogenesis, accounts for 40% of chronic renal failure cases in infants and children, requiring dialysis or transplantation (42). While monogenetic renal dysgenesis syndromes continue to be unraveled, a sizable proportion of cases are sporadic and considered polygenic traits or a result of gene-environment interactions. We recently developed and characterized a unique mouse model of renal dysgenesis that is produced by defined gene-environment interactions. This animal model established that the bradykinin B2 receptor (B2R) is required for normal renal development under conditions of fetal stress (16). Thus, if salt loading is initiated during embryogenesis, the B2R-null progeny acquire an aberrant renal phenotype that is evident histologically on embryonic day 16 (E16), shortly after the onset of metanephric B2R gene expression. The renal phenotype consists of collecting duct dysgenesis, cyst formation, and stromal expansion. In contrast, B2R mutant mice maintained on normal-sodium intake or salt-loaded wild-type mice do not develop kidney abnormalities (16). Postnatal follow-up of gestational salt-stressed B2R–/– mice revealed early-onset salt-sensitive hypertension (9), which was followed by progression to salt-losing nephropathy by 1 yr of age (23). By 18 mo of age, surviving B2R–/– mice show downregulation of the renin-angiotensin system, experience salt wasting, and acquire renal mesenchymal tumors (23).
To gain insights into the underlying susceptibility to renal dysgenesis, we performed a targeted proteomic screen on kidneys from B2R–/– and B2R+/+ pups born to mothers subjected to high-salt intake during gestation. The results revealed that E-cadherin, a key cell adhesion molecule in epithelial tissues, is one of several epithelial proteins that are differentially repressed in dysplastic kidneys. In contrast, dysplastic kidneys expressed higher levels of the checkpoint kinase Chk1. In addition to phosphorylation of Cdc25 and regulation of G2-M transition (21), Chk1 phosphorylates the tumor suppressor protein p53 on serine residue 20 (48). Ser20 phosphorylation is believed to prevent MDM2 interaction with the NH2 terminus of p53 and to stabilize p53 (1). Some studies have shown an association between downregulation of E-cadherin protein expression and alterations of the p53 protein (p53 gene mutation or accumulation of p53) in tumor samples from patients with breast carcinomas (19). It has also been suggested that E-cadherin repression is a necessary step in the pathways leading to apoptosis (3, 6, 19, 24, 50), e.g., in response to DNA damage or ATP depletion. Furthermore, downregulation of E-cadherin is required for execution of morphogenetic movements and epithelial cell migration during embryogenesis (12, 32, 40, 53). Finally, a recent study has shown that p53 stimulates cell migration (45), raising a possible negative link between p53- and cadherin-mediated cell adhesion during development. Collectively, these findings prompted us to explore the potential role of p53 in mediating downregulation of E-cadherin gene expression in renal dysgenesis. The results indicate that repression of endogenous E-cadherin gene expression depends on p53 gene dosage and suggest a novel role for p53 in the regulation of E-cadherin gene transcription.
METHODS
Targeted proteomics of normal and abnormal kidneys. B2R-null mice have been described previously (5, 16, 23). Pairs of male and female B2R+/+ and –/– mice were placed on high-salt (5% NaCl) isocaloric chow (Harlan Laboratories, Madison, WI) 1 day before mating. Pregnant +/+ and –/– mice were continued on their diets for the duration of gestation. The animal study protocol was in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee.
Kidneys from B2R–/– (n = 32) and wild-type +/+ (n = 29) pups were harvested at 1–3 days of postnatal age and kept at –80°C. Approximately 100 mg pooled kidney tissue from each group were homogenized in 1.7 ml boiling lysis buffer (10 mM Tris·HCl, pH 7.4, 1 mM sodium orthovanadate, 1% SDS) using a polytron at full speed for 15 s. Protein extracts were subjected to Western array analysis as per manufacturer's protocol (BD Transduction). All the primary antibodies were mouse monoclonal, so only one secondary antibody was needed. The membrane was then washed and developed with chemiluminescence using SuperSignal West Pico (Pierce). Significant changes in protein expression were defined as a change of 1.5-fold or greater observed in two separate experiments, each performed in duplicate.
Genetic crossing of B2R–/– and p53+/– mice. Breeding pairs of p53+/– mice on a C57BL6 background were obtained from the Jackson Laboratories (Bar Harbor, ME). Crossing B2R–/– with p53+/– mice generated B2R+/–;p53+/– (F1), which were subsequently mated, and the females were placed on a high-salt diet during pregnancy. The F2 progeny consisted of wild-type, compound heterozygous, and homozygous null genotypes. PCR genotyping was performed on tail genomic DNA according to protocols established by the Jackson Laboratories.
Plasmids. Plasmid pCMV-p53 expresses a wild-type p53 protein from the cytomegalovirus promoter-enhancer in the pCG expression vector. pCMVp53E258K encodes a DNA-binding mutant p53. Human E-cadherin promoter-luciferase constructs (–1359, –368, and –37) were previously described (22, 25). Mutagenesis of the p53 site was performed by the QuickChange mutagenesis system (Stratagene, La Jolla, CA) following the manufacturer's recommendations. The primer sequence (forward) used for mutagenesis is 5'-GTCAGTTCAGACTACATCCCGCTACATCCCGGCCCGAC. All constructs were sequenced to verify the sequence by automated DNA sequencing (model 373A, Applied Biosystems).
Cell culture and transfection. Mouse inner medullary collecting duct cells (IMCD3) were maintained in DMEM/F-12 containing 10% FBS (GIBCO BRL) at 37°C in a humidified incubator with 5% CO2. H1299 (p53-null lung carcinoma) cells were maintained in DMEM/high glucose containing 4,500 mg/l D-glucose, L-glutamine, and 25 mM HEPES buffer, no sodium pyruvate, and 10% FBS. Isogenic HCT116 (human colon carcinoma) p53–/– and p53+/+ cells were cultured in McCoy's 5A modified medium supplemented with 10% FBS. Cells were plated in duplicate in six-well plates at 5–7 x 105 cells/well in DMEM containing 10% FBS 1 day before transfection. Transfections were performed using LipofectAMINE PLUS Reagent (GIBCO BRL) according to the manufacturer's recommendations, as described (46). In experiments utilizing trichostatin A (TSA), cells were treated with TSA (100 ng/ml) or vehicle (DMSO) 2 h after plating, transfected the following day, and cell extracts were harvested 24 h later.
Preparation of nuclear extracts and EMSA. EMSA was performed as described (41). The oligonucleotide sequences used in the gel shift assays were as follows: the high-affinilty p53-binding site (P1) from the rat B2R gene promoter, 5'-ggggGGAGGTGCCCAGGAGAGTGAtgaca-3' (47); the p53-consensus sequence in the human E-cadherin promoter (7); wild-type (wtE-cad), 5'-cagACTCCAGCCCGCTCCAGCCCggc-3' (nucleotide position 1072; accession NM009864); and mutant (mtE-cad): cagACTACATCCCGCTACATCCCggc.
Immunoblotting.. Western blot analysis was performed as previously described (23) using a polyclonal E-cadherin antibody diluted 1:500.
-Irradiation, RNA extraction, and RT-PCR. Confluent HCT116 p53+/+ and p53–/– cells were subjected to irradiation (8-Gy), and total RNA was isolated at 0, 2, and 6 h after irradiation using the TRIzol reagent (Invitrogen). First-strand cDNA was synthesized using 5 μg of total cellular RNA as a template, 0.1 μg random hexamers (Invitrogen), 4 μl of 5x first-strand buffer (Invitrogen), 2 μl of 10 mM dithiothreitol, 0.5 μl of 20 mM dNTPs, and 1 μl of Superscript II (Invitrogen) in a volume of 20 μl. For E-cadherin, 8 μl of cDNA template, 5 μl 10x PCR buffer, 1.5 μl of 50 mM MgCl2, 0.5 μl of 20 mM dNTPs, 1 μl of 25 pmol forward and reverse primers, and 1 μl (5 U) of Taq polymerase (Invitrogen) were applied to the following PCR program: 4 min at 95°C, 40 s at 95°C, 30 s at 53°C, 1 min at 72°C, and final extension 7 min at 72°C for 35 cycles. After amplification, 15 μl of each PCR reaction mixture were electrophoresed through a 1% agarose gel with ethidium bromide (0.5 μg/ml). The gel was scanned with ultraviolet illumination using digital imaging and analysis (Alpha Innotech). For GAPDH, the same protocol was used except for 2 μl of cDNA template, annealing temperature of 55°C, and 30 cycles. The E-cadherin primers are forward (exon 12): 5'-TTCCTCCCAATACATCTCCCTTCACAGCAG and 5'-reverse (exon 14): 5'-CGAAGAAACAGCAAGAGCAGCAGAATCAGA. The GAPDH primers are forward 5'-AATGCATCCTGCACCACCAA and reverse 5'-GTAGCCATATTCATTGTCATA. The expected sizes of the E-cadherin and GAPDH PCR products are 339 and 515 bp, respectively.
Chromatin immunoprecipitation. Chromatin immunoprecipitation (ChIP) assays were performed using reagents from Upstate Biotechnology. p53 Null H1299 cells were plated in 6-well plates at a density of 4 x 105 cells/well and cultured in DMEM/D-glucose, L-glutamine, and 25 mM HEPES buffer and 10% FBS. The following day, the cells were treated with TSA or its vehicle (DMSO; 100 ng/ml). After 18 h, the cells were either transfected with pCMV-p53 plasmid DNA (100 ng) or mock transfected. Twenty-four hours after transfection, cells were treated with a 1% formaldehyde solution in PBS for cross-linking for 10 min at 37°C. Reaction was quenched by addition of 0.125 M glycine. Cells were rinsed twice in ice-cold 1x PBS and lysed in SDS lysis buffer with protease inhibitors. DNA was sheared by sonication and diluted 10-fold in ChIP dilution buffer. Immunoprecipitation was done with anti-acetylated histone H4 (Upstate Biotechnology) antibody (1:150 dilution) overnight at 4°C. DNA-protein-antibody complexes were captured on protein A-conjugated agarose beads. After washing and elution of the complexes from the beads, DNA-protein cross-links were reversed at 65°C overnight. Immunoprecipitated DNA was ethanol precipitated after proteins were removed by phenol-chloroform-isoamyl alcohol extraction after proteinase K treatment and used for PCR of the human E-cadherin gene in the region flanking the p53-binding site. Sequences of the primers used for 30 cycles of PCR are as follows: forward primer 5'-TAGACCCTAGCAACTCCAG with reverse primer 5'-CTGAACTGACTTCCGCAAGC to amplify the endogenous human E-cadherin DNA (size of amplicon is 241 bp).
RESULTS
Differentially expressed proteins in dysplastic kidneys. B2R-knockout mice are phenotypically normal, but, when B2R–/– mice are exposed to high-salt stress in utero, a kidney-specific developmental defect is induced. To gain insights into the pathogenesis of the renal lesion, we performed a targeted proteomic screen. Of the 724 proteins surveyed, 22 (3%) showed a reproducible change of at least 1.5-fold in dysplastic (i.e., salt-stressed B2R–/–) compared with normal (i.e., salt-stressed B2R+/+) kidneys (Tables 1 and 2). Of these, 14 proteins were downregulated. Conventional Western blotting confirmed the results in 5 randomly selected proteins (PCNA, Cul-2, E-cadherin, eNOS, HNF-1) (data not shown).
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Dysplastic kidneys have lower levels of E-cadherin and higher levels of Chk1 and Ser20-p53. Dysplastic kidneys expressed higher levels of the checkpoint kinase Chk1 (+19.5-fold) and c-Jun and JNK1, components of the Jun kinase signaling pathway (Table 2). Representative Western arrays are shown in Figs. 1 and 2. Chk1 and JNK are activated by stressful stimuli, and both kinases phosphorylate p53 (20, 48). Chk1 phosphorylates p53 on Ser20. This modification stabilizes p53. Western blot analysis of nuclear extracts prepared from salt-stressed B2R–/– and +/+ pups revealed a 2.5-fold higher level of Ser20-p-p53 in B2R–/– than +/+ kidneys (Fig. 3A), indicating activation of p53 in the dysplastic kidneys.
p53 Gene dosage reduction restores kidney E-cadherin gene expression and nephron differentiation in B2R–/– mice. To determine whether there is a functional link between p53 and E-cadherin in vivo, we crossed p53+/– with B2R–/– mice to generate p53+/–;B2R+/– mice, which were then mated and placed on high salt during pregnancy (Fig. 3B). Kidney E-cadherin/GAPDH mRNA levels were 3.3-fold lower in kidneys of salt-stressed B2R–/–;p53+/+ mice than in littermates with other genotypes (Fig. 3C). Importantly, the p53 gene dosage reduction (i.e., B2R–/–;p53+/–) was sufficient to restore E-cadherin mRNA levels. These results strongly argue in favor of the notion that p53 downregulates E-cadherin gene expression in vivo.
We next examined the effect of p53 gene dosage reduction on the renal phenotype of salt-stressed B2R–/– mice. As previously described, salt-stressed B2R–/–;p53+/+ pups have renal dysgenesis, including cyst formation and dedifferentiation of renal epithelia (Fig. 4, C and D). In contrast, the renal architecture in B2R–/–;p53+/– littermates was normal (Fig. 4, A and B), indicating that p53 activation plays a role in the pathogenesis of the renal dysgenesis.
p53 Represses E-cadherin promoter activity. Sequence analysis of the human E-cadherin gene promoter (7) for putative transcription factor binding sites revealed the presence of a putative p53 binding site with sequence similarity to the p53 consensus sequence RRRC(A/T)(T/A)GYYY located in exon 1, 50 nucleotides downstream of the transcription initiation site (underlined sequence in rectangle, Fig. 5A). Using EMSA, we tested the binding of bacterially produced full-length human p53 to this DNA element (in the presence of activating antibody Pab421). As shown in Fig. 5B, p53 binds with relatively high affinity to the radiolabeled E-cadherin oligoduplex but does not bind a mutant oligoduplex (compare lanes 1 and 3). The binding intensity of p53 to the E-cadherin DNA element is lower than that of a high-affinity p53 binding site in the rat B2R gene (47) (compare lanes 1 and 4). Recombinant p73, a p53-related family member, also binds the E-cadherin p53 binding-site, albeit at a lower affinity than p53 (compare lanes 1 and 2).
To determine the effect of p53 on E-cadherin transcription, luciferase reporter constructs driven by various lengths of the human E-cadherin promoter were transfected with or without pCMV-p53 into either H1299 (p53-null lung carcinoma cells) or mIMCD3 (immortalized mouse inner medullary collecting duct cells). As shown in Fig. 5C, p53 dose dependently repressed E-cadherin promoter activity up to 10-fold in H1299 cells. Introduction of p53 into IMCD3 cells caused a similar inhibitory effect on E-cadherin promoter activity (Fig. 5D), although baseline E-cadherin promoter activity was 50- to 100-fold higher in IMCD3 than H1299 cells (Fig. 5, C and D). Importantly, p53 repressed all three E-cadherin-luciferase constructs (position –1359/+125, –368/+125, and –37/+125), consistent with the location of the p53-binding site (position +50). Western blots of extracts from p53-transfected cells revealed that p53 represses endogenous E-cadherin expression (Fig. 5D, inset). Of note is that the p53 homolog p73 was also capable of repressing the three promoter constructs in H1299 cells but to a lower extent (50% that of p53) (data not shown).
As mentioned above, a p53-binding site (ACTCCAGCCCGCTCCAGCCC) is present at nucleotide position +50 of the human E-cadherin promoter, and mutagenesis of this site (underlined bases) eliminates p53-binding (Fig. 5, A and B). We evaluated the functional consequences of these mutations on p53-mediated repression of the E-cadherin promoter in H1299 cells. Figure 6C shows that increasing amounts of p53 repressed the E-cadherin promoter in a dose-dependent manner. The mutant E-cadherin promoter tended to have a higher basal promoter activity than the wild-type promoter, although the difference did not reach statistical significance. In addition, the mutant promoter was less amenable to repression at a 50-ng p53 dose than the wild-type promoter (70 vs. 35%, P < 0.05) (Fig. 6C). Importantly, a mutant of p53 (p53E258K), which is unable to bind DNA, failed to repress the E-cadherin promoter despite equal cellular expression (Fig. 6, B and C). These results indicate that DNA binding is required for p53-mediated repression of the E-cadherin promoter and that the p53-binding site at nucleotide position +50 in the E-cadherin promoter is required, although not sufficient for p53-mediated repression.
p53-Mediated repression of endogenous E-cadherin gene expression in response to DNA damage. To assess the contribution of endogenous p53 to E-cadherin gene regulation, we compared E-cadherin gene expression in -irradiated (IR) HCT116 p53+/+ and p53–/– cells by RT-PCR. As expected, IR induced Ser20 phosphorylation of p53 in HCT116 p53+/+ cells (Fig. 7A). In contrast, IR suppressed E-cadherin mRNA levels (factored for GAPDH) in p53+/+ by as much as 70% compared with 5–10% in p53–/– cells (Fig. 7B). These findings imply that repression of endogenous E-cadherin gene expression in response to DNA damage is p53 dependent.
p53-Mediated repression of E-cadherin is trichostatin sensitive. p53 Represses gene transcription via multiple mechanisms, including recruitment of histone deacetylase (HDAC) complexes to the target promoter (28, 37). To test this possibility, H1299 cells were treated with the selective HDAC inhibitor TSA 18 h before transfection with the p53 expression vector and E-cadherin-luciferase constructs. TSA increased basal E-cadherin promoter activity (Fig. 8A), suggesting that HDACs participate in the physiological repression of the E-cadherin promoter. Importantly, TSA attenuated p53-mediated repression in all test constructs (Fig. 8A), suggesting that p53 represses the E-cadherin promoter, at least partly, via HDAC-dependent mechanisms.
To correlate the magnitude of histone acetylation in the E-cadherin promoter with the observed transcriptional responses, we performed ChIP analysis in p53-transfected H1299 cells in the presence or absence of TSA treatment. The results revealed that p53 decreased basal E-cadherin promoter H4 acetylation (Fig. 8B), whereas TSA treatment restored promoter-acetylated H4 levels to control levels. Control input DNA samples showed no differences between the treatment groups. In addition, control samples processed with omission of the immunoprecipitation step did not reveal an amplification product (Fig. 8B). These findings are consistent with the functional transcriptional responses whereby TSA restored E-cadherin promoter activity (Fig. 8A).
DISCUSSION
The present study explores the factors underlying aberrant gene regulation and morphogenesis in a gene-environment interaction model of renal dysgenesis. Two lines of independent evidence support the conclusion that p53 mediates repression of E-cadherin gene expression in vivo. First, DNA damage induced by IR is associated with reductions in endogenous E-cadherin mRNA levels in p53+/+ but not p53–/– cells. Second, genetic crosses between B2R–/– and p53+/– resulting in reduced p53 gene dosage restore kidney E-cadherin gene expression. In addition to E-cadherin, the dysplastic kidneys expressed lower levels of other epithelial differentiation genes, including Dlg, HNF-1, and cullin-2. Interestingly, mutations of these genes cause dedifferentiation and cancer (39, 43, 56), which may explain, at least in part, the susceptibility of salt-stressed B2R-null mice to tumorigenesis with aging (23). The mechanisms leading to the downregulation of these tumor suppressors in dysplastic B2R–/– kidneys will be the subject of future investigations.
E-cadherin is a calcium-dependent cell adhesion molecule, which forms a molecular complex with -catenin, plakoglobin, and p120ctn. Through this complex, E-cadherin is connected to the actin cytoskeleton (32, 51, 57). The E-cadherin-catenin adhesion complex is essential for early embryonic development, since E-cadherin-deficient embryos die at the blastocyst stage (29, 44). In addition, E-cadherin has tumor suppressor functions, and mutations affecting its synthesis or transcription occur in many human tumors (4, 7, 10). There is also evidence that E-cadherin functions as a survival factor during terminal epithelial differentiation, since knockdown of E-cadherin expression in the lactating mammary gland induces epithelial cell apoptosis (6). Collectively, the available data suggest that cellular E-cadherin levels must be finely tuned to achieve optimal epithelial sheet adhesion, migration, polarization, and cytoskeleton integrity (40, 55).
p53 Has dual roles as a transcriptional activator or repressor. The consensus p53-response element consists of two copies of the inverted repeats (RRRCA/TT/AGYYY), separated by 0–13 bp (8, 17, 27, 54). The mechanisms of p53-mediated transcriptional repression are far less well understood than those of activation. Recruitment of HDACs, displacement of activators, or interactions with the basal transcription machinery are potential mechanisms of p53-mediated repression (18, 26, 30, 33, 34). The results of this study suggest that direct binding of p53 to the E-cadherin promoter is required, although not sufficient for p53-mediated repression since 1) a mutant p53 that cannot bind DNA did not repress the E-cadherin promoter and 2) mutation of the p53-binding site attenuated but did not eliminate p53-induced repression. It is possible that p53 represses E-cadherin transcription partly by interacting with and squelching transcriptional activators (18). Another mechanism by which p53 could influence E-cadherin expression is through binding with Smad2 (11). Smad2 represses E-cadherin expression, leading to epithelial-mesenchymal transition (31).
Acetylation of histone NH2 termini by histone acetylases (e.g., CBP/p300) alters chromatin conformation, allowing transcriptional regulators better access to DNA-regulatory elements (2). The balance between histone acetylase and HDAC activity is an important determinant of gene transcription. Previous studies have demonstrated that p53 represses certain genes via recruitment of HDAC-containing repressor complexes (36). Experiments that utilized TSA to inhibit endogenous HDAC activity revealed increased baseline E-cadherin promoter activity and attenuation of p53-mediated repression. In addition, ChIP assays revealed that TSA treatment of p53-transfected cells restores histone H4 acetylation in the E-cadherin core promoter region. One of our future goals is to adapt the ChIP technique to embryonic tissues to map occupancy of p53-HDAC complexes on the endogenous E-cadherin gene. Reestablishing expression of E-cadherin (and other differentiation genes) by HDAC inhibitors may offer a therapeutic strategy in experimental renal dysgenesis, similar to what has been described in cancer (35, 52).
The mechanisms leading to activated p53 in this model of renal dysgenesis are not completely clear. Our proteomic screen has shown that although Chk1 is upregulated 19.5- fold, P-Ser20-p53 is only increased 2.5-fold in dysplastic B2R–/– kidneys. The difference may represent differential antibody sensitivity or, more likely, the fact that full p53 stabilization requires additional posttranslational modifications including NH2-terminal phosphoprylation and COOH-terminal acetylation (38). IR-induced Ser20 phosphorylation is mediated via Chk1 activation (48). At first glance, these data suggest that the pathogenesis of renal dysgenesis mimics that of genotoxic stress. However, DNA damage-induced Chk1 activation results from phosphorylation by upstream kinases (e.g., ATM/ATR) rather than increased protein levels, as is observed in dysplastic kidneys. Moreover, p53 is known to repress transcription of the human Chk1 gene (13). These findings suggest that DNA damage as well as other pathways leading to the activation of p53 may be involved in the susceptibility of B2R-null mice to renal dysgenesis. It is unknown yet whether maternal high salt crosses the placenta to reach the fetal circulation. Studies conducted in cultured inner medullary collecting duct cells have shown that high salt induces DNA strand breaks and activates p53 (15).
The full biological significance of p53-mediated repression of E-cadherin gene expression is likely to be context dependent. Since E-cadherin function is required for cell survival (3, 6, 14), it is conceivable that p53-mediated repression of E-cadherin is a mechanism to promote cell death (19). Preliminary studies in our laboratory indicate that B2R-null dysplastic kidneys exhibit widespread apoptosis and that rescue of renal dysgenesis by reduction of p53 gene dosage (shown in the current study) is accompanied by restriction of cell death (Fan H and El-Dahr SS, unpublished observations). In addition, downregulation of E-cadherin by p53 can promote epithelial-mesenchymal transition (49), which may explain expansion of the mesenchymal stroma typically observed in dysplastic kidneys.
GRANTS
These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-56264 and DK-62250, the Tulane Renal and Hypertension Center of Excellence, and the American Heart Association, Southeast Affiliate.
ACKNOWLEDGMENTS
We thank Drs. Eric Fearon and Bert Vogelstein for providing the E-cadherin promoter constructs and HCT116 cells, respectively.
FOOTNOTES
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.
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ABSTRACT
Gene-environment interactions are implicated in congenital human disorders. Accordingly, there is a pressing need to develop animal models of human disease, which are the product of defined gene-environment interactions. Previously, our laboratory demonstrated that gestational salt stress of bradykinin B2 receptor (B2R)-null mice induces renal dysgenesis and early death of the offspring (El-Dahr SS, Harrison-Bernard LM, Dipp S, Yosipiv IV, and Meleg-Smith S. Physiol Genomics 3: 121–131, 2000). In contrast, salt-stressed B2R +/+ or +/– littermates have normal development. The present study investigates the mechanisms underlying the susceptibility of B2R-null mice to renal dysgenesis. Proteomic and conventional Western blot screens identified E-cadherin among the differentially repressed proteins in B2R–/– kidneys, whereas the checkpoint kinase Chk1 and its substrate P-Ser20 p53 were induced. We tested the hypothesis that p53 mediates repression of E-cadherin gene expression and is causally linked to the renal dysgenesis. Genetic crosses between B2R –/– and p53+/– mice revealed that germline reduction of p53 gene dosage rescues B2R–/– mice from renal dysgenesis and restores kidney E-cadherin gene expression. Furthermore, -irradiation induces repression of E-cadherin gene expression in p53+/+ but not –/– cells. In transient transfection assays, p53 repressed human E-cadherin promoter-driven reporter activity, whereas a mutant p53, which cannot bind DNA, did not. Functional promoter analysis indicated the presence of a p53-responsive element in exon 1, which partially mediates p53-induced repression. Chromatin immunoprecipitation assays revealed that p53 inhibits histone acetylation of the E-cadherin promoter. Treatment with a histone deacetylase inhibitor reversed both p53-mediated promoter repression and deacetylation. In conclusion, this study demonstrates that gene-environment interactions cooperate to induce congenital defects through p53 activation.
bradykinin B2 receptor; knockout mice; checkpoint kinase; histone acetylation
CONGENITAL RENAL DYSGENESIS, leading to hypoplasia, dysplasia, and cystogenesis, accounts for 40% of chronic renal failure cases in infants and children, requiring dialysis or transplantation (42). While monogenetic renal dysgenesis syndromes continue to be unraveled, a sizable proportion of cases are sporadic and considered polygenic traits or a result of gene-environment interactions. We recently developed and characterized a unique mouse model of renal dysgenesis that is produced by defined gene-environment interactions. This animal model established that the bradykinin B2 receptor (B2R) is required for normal renal development under conditions of fetal stress (16). Thus, if salt loading is initiated during embryogenesis, the B2R-null progeny acquire an aberrant renal phenotype that is evident histologically on embryonic day 16 (E16), shortly after the onset of metanephric B2R gene expression. The renal phenotype consists of collecting duct dysgenesis, cyst formation, and stromal expansion. In contrast, B2R mutant mice maintained on normal-sodium intake or salt-loaded wild-type mice do not develop kidney abnormalities (16). Postnatal follow-up of gestational salt-stressed B2R–/– mice revealed early-onset salt-sensitive hypertension (9), which was followed by progression to salt-losing nephropathy by 1 yr of age (23). By 18 mo of age, surviving B2R–/– mice show downregulation of the renin-angiotensin system, experience salt wasting, and acquire renal mesenchymal tumors (23).
To gain insights into the underlying susceptibility to renal dysgenesis, we performed a targeted proteomic screen on kidneys from B2R–/– and B2R+/+ pups born to mothers subjected to high-salt intake during gestation. The results revealed that E-cadherin, a key cell adhesion molecule in epithelial tissues, is one of several epithelial proteins that are differentially repressed in dysplastic kidneys. In contrast, dysplastic kidneys expressed higher levels of the checkpoint kinase Chk1. In addition to phosphorylation of Cdc25 and regulation of G2-M transition (21), Chk1 phosphorylates the tumor suppressor protein p53 on serine residue 20 (48). Ser20 phosphorylation is believed to prevent MDM2 interaction with the NH2 terminus of p53 and to stabilize p53 (1). Some studies have shown an association between downregulation of E-cadherin protein expression and alterations of the p53 protein (p53 gene mutation or accumulation of p53) in tumor samples from patients with breast carcinomas (19). It has also been suggested that E-cadherin repression is a necessary step in the pathways leading to apoptosis (3, 6, 19, 24, 50), e.g., in response to DNA damage or ATP depletion. Furthermore, downregulation of E-cadherin is required for execution of morphogenetic movements and epithelial cell migration during embryogenesis (12, 32, 40, 53). Finally, a recent study has shown that p53 stimulates cell migration (45), raising a possible negative link between p53- and cadherin-mediated cell adhesion during development. Collectively, these findings prompted us to explore the potential role of p53 in mediating downregulation of E-cadherin gene expression in renal dysgenesis. The results indicate that repression of endogenous E-cadherin gene expression depends on p53 gene dosage and suggest a novel role for p53 in the regulation of E-cadherin gene transcription.
METHODS
Targeted proteomics of normal and abnormal kidneys. B2R-null mice have been described previously (5, 16, 23). Pairs of male and female B2R+/+ and –/– mice were placed on high-salt (5% NaCl) isocaloric chow (Harlan Laboratories, Madison, WI) 1 day before mating. Pregnant +/+ and –/– mice were continued on their diets for the duration of gestation. The animal study protocol was in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee.
Kidneys from B2R–/– (n = 32) and wild-type +/+ (n = 29) pups were harvested at 1–3 days of postnatal age and kept at –80°C. Approximately 100 mg pooled kidney tissue from each group were homogenized in 1.7 ml boiling lysis buffer (10 mM Tris·HCl, pH 7.4, 1 mM sodium orthovanadate, 1% SDS) using a polytron at full speed for 15 s. Protein extracts were subjected to Western array analysis as per manufacturer's protocol (BD Transduction). All the primary antibodies were mouse monoclonal, so only one secondary antibody was needed. The membrane was then washed and developed with chemiluminescence using SuperSignal West Pico (Pierce). Significant changes in protein expression were defined as a change of 1.5-fold or greater observed in two separate experiments, each performed in duplicate.
Genetic crossing of B2R–/– and p53+/– mice. Breeding pairs of p53+/– mice on a C57BL6 background were obtained from the Jackson Laboratories (Bar Harbor, ME). Crossing B2R–/– with p53+/– mice generated B2R+/–;p53+/– (F1), which were subsequently mated, and the females were placed on a high-salt diet during pregnancy. The F2 progeny consisted of wild-type, compound heterozygous, and homozygous null genotypes. PCR genotyping was performed on tail genomic DNA according to protocols established by the Jackson Laboratories.
Plasmids. Plasmid pCMV-p53 expresses a wild-type p53 protein from the cytomegalovirus promoter-enhancer in the pCG expression vector. pCMVp53E258K encodes a DNA-binding mutant p53. Human E-cadherin promoter-luciferase constructs (–1359, –368, and –37) were previously described (22, 25). Mutagenesis of the p53 site was performed by the QuickChange mutagenesis system (Stratagene, La Jolla, CA) following the manufacturer's recommendations. The primer sequence (forward) used for mutagenesis is 5'-GTCAGTTCAGACTACATCCCGCTACATCCCGGCCCGAC. All constructs were sequenced to verify the sequence by automated DNA sequencing (model 373A, Applied Biosystems).
Cell culture and transfection. Mouse inner medullary collecting duct cells (IMCD3) were maintained in DMEM/F-12 containing 10% FBS (GIBCO BRL) at 37°C in a humidified incubator with 5% CO2. H1299 (p53-null lung carcinoma) cells were maintained in DMEM/high glucose containing 4,500 mg/l D-glucose, L-glutamine, and 25 mM HEPES buffer, no sodium pyruvate, and 10% FBS. Isogenic HCT116 (human colon carcinoma) p53–/– and p53+/+ cells were cultured in McCoy's 5A modified medium supplemented with 10% FBS. Cells were plated in duplicate in six-well plates at 5–7 x 105 cells/well in DMEM containing 10% FBS 1 day before transfection. Transfections were performed using LipofectAMINE PLUS Reagent (GIBCO BRL) according to the manufacturer's recommendations, as described (46). In experiments utilizing trichostatin A (TSA), cells were treated with TSA (100 ng/ml) or vehicle (DMSO) 2 h after plating, transfected the following day, and cell extracts were harvested 24 h later.
Preparation of nuclear extracts and EMSA. EMSA was performed as described (41). The oligonucleotide sequences used in the gel shift assays were as follows: the high-affinilty p53-binding site (P1) from the rat B2R gene promoter, 5'-ggggGGAGGTGCCCAGGAGAGTGAtgaca-3' (47); the p53-consensus sequence in the human E-cadherin promoter (7); wild-type (wtE-cad), 5'-cagACTCCAGCCCGCTCCAGCCCggc-3' (nucleotide position 1072; accession NM009864); and mutant (mtE-cad): cagACTACATCCCGCTACATCCCggc.
Immunoblotting.. Western blot analysis was performed as previously described (23) using a polyclonal E-cadherin antibody diluted 1:500.
-Irradiation, RNA extraction, and RT-PCR. Confluent HCT116 p53+/+ and p53–/– cells were subjected to irradiation (8-Gy), and total RNA was isolated at 0, 2, and 6 h after irradiation using the TRIzol reagent (Invitrogen). First-strand cDNA was synthesized using 5 μg of total cellular RNA as a template, 0.1 μg random hexamers (Invitrogen), 4 μl of 5x first-strand buffer (Invitrogen), 2 μl of 10 mM dithiothreitol, 0.5 μl of 20 mM dNTPs, and 1 μl of Superscript II (Invitrogen) in a volume of 20 μl. For E-cadherin, 8 μl of cDNA template, 5 μl 10x PCR buffer, 1.5 μl of 50 mM MgCl2, 0.5 μl of 20 mM dNTPs, 1 μl of 25 pmol forward and reverse primers, and 1 μl (5 U) of Taq polymerase (Invitrogen) were applied to the following PCR program: 4 min at 95°C, 40 s at 95°C, 30 s at 53°C, 1 min at 72°C, and final extension 7 min at 72°C for 35 cycles. After amplification, 15 μl of each PCR reaction mixture were electrophoresed through a 1% agarose gel with ethidium bromide (0.5 μg/ml). The gel was scanned with ultraviolet illumination using digital imaging and analysis (Alpha Innotech). For GAPDH, the same protocol was used except for 2 μl of cDNA template, annealing temperature of 55°C, and 30 cycles. The E-cadherin primers are forward (exon 12): 5'-TTCCTCCCAATACATCTCCCTTCACAGCAG and 5'-reverse (exon 14): 5'-CGAAGAAACAGCAAGAGCAGCAGAATCAGA. The GAPDH primers are forward 5'-AATGCATCCTGCACCACCAA and reverse 5'-GTAGCCATATTCATTGTCATA. The expected sizes of the E-cadherin and GAPDH PCR products are 339 and 515 bp, respectively.
Chromatin immunoprecipitation. Chromatin immunoprecipitation (ChIP) assays were performed using reagents from Upstate Biotechnology. p53 Null H1299 cells were plated in 6-well plates at a density of 4 x 105 cells/well and cultured in DMEM/D-glucose, L-glutamine, and 25 mM HEPES buffer and 10% FBS. The following day, the cells were treated with TSA or its vehicle (DMSO; 100 ng/ml). After 18 h, the cells were either transfected with pCMV-p53 plasmid DNA (100 ng) or mock transfected. Twenty-four hours after transfection, cells were treated with a 1% formaldehyde solution in PBS for cross-linking for 10 min at 37°C. Reaction was quenched by addition of 0.125 M glycine. Cells were rinsed twice in ice-cold 1x PBS and lysed in SDS lysis buffer with protease inhibitors. DNA was sheared by sonication and diluted 10-fold in ChIP dilution buffer. Immunoprecipitation was done with anti-acetylated histone H4 (Upstate Biotechnology) antibody (1:150 dilution) overnight at 4°C. DNA-protein-antibody complexes were captured on protein A-conjugated agarose beads. After washing and elution of the complexes from the beads, DNA-protein cross-links were reversed at 65°C overnight. Immunoprecipitated DNA was ethanol precipitated after proteins were removed by phenol-chloroform-isoamyl alcohol extraction after proteinase K treatment and used for PCR of the human E-cadherin gene in the region flanking the p53-binding site. Sequences of the primers used for 30 cycles of PCR are as follows: forward primer 5'-TAGACCCTAGCAACTCCAG with reverse primer 5'-CTGAACTGACTTCCGCAAGC to amplify the endogenous human E-cadherin DNA (size of amplicon is 241 bp).
RESULTS
Differentially expressed proteins in dysplastic kidneys. B2R-knockout mice are phenotypically normal, but, when B2R–/– mice are exposed to high-salt stress in utero, a kidney-specific developmental defect is induced. To gain insights into the pathogenesis of the renal lesion, we performed a targeted proteomic screen. Of the 724 proteins surveyed, 22 (3%) showed a reproducible change of at least 1.5-fold in dysplastic (i.e., salt-stressed B2R–/–) compared with normal (i.e., salt-stressed B2R+/+) kidneys (Tables 1 and 2). Of these, 14 proteins were downregulated. Conventional Western blotting confirmed the results in 5 randomly selected proteins (PCNA, Cul-2, E-cadherin, eNOS, HNF-1) (data not shown).
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Dysplastic kidneys have lower levels of E-cadherin and higher levels of Chk1 and Ser20-p53. Dysplastic kidneys expressed higher levels of the checkpoint kinase Chk1 (+19.5-fold) and c-Jun and JNK1, components of the Jun kinase signaling pathway (Table 2). Representative Western arrays are shown in Figs. 1 and 2. Chk1 and JNK are activated by stressful stimuli, and both kinases phosphorylate p53 (20, 48). Chk1 phosphorylates p53 on Ser20. This modification stabilizes p53. Western blot analysis of nuclear extracts prepared from salt-stressed B2R–/– and +/+ pups revealed a 2.5-fold higher level of Ser20-p-p53 in B2R–/– than +/+ kidneys (Fig. 3A), indicating activation of p53 in the dysplastic kidneys.
p53 Gene dosage reduction restores kidney E-cadherin gene expression and nephron differentiation in B2R–/– mice. To determine whether there is a functional link between p53 and E-cadherin in vivo, we crossed p53+/– with B2R–/– mice to generate p53+/–;B2R+/– mice, which were then mated and placed on high salt during pregnancy (Fig. 3B). Kidney E-cadherin/GAPDH mRNA levels were 3.3-fold lower in kidneys of salt-stressed B2R–/–;p53+/+ mice than in littermates with other genotypes (Fig. 3C). Importantly, the p53 gene dosage reduction (i.e., B2R–/–;p53+/–) was sufficient to restore E-cadherin mRNA levels. These results strongly argue in favor of the notion that p53 downregulates E-cadherin gene expression in vivo.
We next examined the effect of p53 gene dosage reduction on the renal phenotype of salt-stressed B2R–/– mice. As previously described, salt-stressed B2R–/–;p53+/+ pups have renal dysgenesis, including cyst formation and dedifferentiation of renal epithelia (Fig. 4, C and D). In contrast, the renal architecture in B2R–/–;p53+/– littermates was normal (Fig. 4, A and B), indicating that p53 activation plays a role in the pathogenesis of the renal dysgenesis.
p53 Represses E-cadherin promoter activity. Sequence analysis of the human E-cadherin gene promoter (7) for putative transcription factor binding sites revealed the presence of a putative p53 binding site with sequence similarity to the p53 consensus sequence RRRC(A/T)(T/A)GYYY located in exon 1, 50 nucleotides downstream of the transcription initiation site (underlined sequence in rectangle, Fig. 5A). Using EMSA, we tested the binding of bacterially produced full-length human p53 to this DNA element (in the presence of activating antibody Pab421). As shown in Fig. 5B, p53 binds with relatively high affinity to the radiolabeled E-cadherin oligoduplex but does not bind a mutant oligoduplex (compare lanes 1 and 3). The binding intensity of p53 to the E-cadherin DNA element is lower than that of a high-affinity p53 binding site in the rat B2R gene (47) (compare lanes 1 and 4). Recombinant p73, a p53-related family member, also binds the E-cadherin p53 binding-site, albeit at a lower affinity than p53 (compare lanes 1 and 2).
To determine the effect of p53 on E-cadherin transcription, luciferase reporter constructs driven by various lengths of the human E-cadherin promoter were transfected with or without pCMV-p53 into either H1299 (p53-null lung carcinoma cells) or mIMCD3 (immortalized mouse inner medullary collecting duct cells). As shown in Fig. 5C, p53 dose dependently repressed E-cadherin promoter activity up to 10-fold in H1299 cells. Introduction of p53 into IMCD3 cells caused a similar inhibitory effect on E-cadherin promoter activity (Fig. 5D), although baseline E-cadherin promoter activity was 50- to 100-fold higher in IMCD3 than H1299 cells (Fig. 5, C and D). Importantly, p53 repressed all three E-cadherin-luciferase constructs (position –1359/+125, –368/+125, and –37/+125), consistent with the location of the p53-binding site (position +50). Western blots of extracts from p53-transfected cells revealed that p53 represses endogenous E-cadherin expression (Fig. 5D, inset). Of note is that the p53 homolog p73 was also capable of repressing the three promoter constructs in H1299 cells but to a lower extent (50% that of p53) (data not shown).
As mentioned above, a p53-binding site (ACTCCAGCCCGCTCCAGCCC) is present at nucleotide position +50 of the human E-cadherin promoter, and mutagenesis of this site (underlined bases) eliminates p53-binding (Fig. 5, A and B). We evaluated the functional consequences of these mutations on p53-mediated repression of the E-cadherin promoter in H1299 cells. Figure 6C shows that increasing amounts of p53 repressed the E-cadherin promoter in a dose-dependent manner. The mutant E-cadherin promoter tended to have a higher basal promoter activity than the wild-type promoter, although the difference did not reach statistical significance. In addition, the mutant promoter was less amenable to repression at a 50-ng p53 dose than the wild-type promoter (70 vs. 35%, P < 0.05) (Fig. 6C). Importantly, a mutant of p53 (p53E258K), which is unable to bind DNA, failed to repress the E-cadherin promoter despite equal cellular expression (Fig. 6, B and C). These results indicate that DNA binding is required for p53-mediated repression of the E-cadherin promoter and that the p53-binding site at nucleotide position +50 in the E-cadherin promoter is required, although not sufficient for p53-mediated repression.
p53-Mediated repression of endogenous E-cadherin gene expression in response to DNA damage. To assess the contribution of endogenous p53 to E-cadherin gene regulation, we compared E-cadherin gene expression in -irradiated (IR) HCT116 p53+/+ and p53–/– cells by RT-PCR. As expected, IR induced Ser20 phosphorylation of p53 in HCT116 p53+/+ cells (Fig. 7A). In contrast, IR suppressed E-cadherin mRNA levels (factored for GAPDH) in p53+/+ by as much as 70% compared with 5–10% in p53–/– cells (Fig. 7B). These findings imply that repression of endogenous E-cadherin gene expression in response to DNA damage is p53 dependent.
p53-Mediated repression of E-cadherin is trichostatin sensitive. p53 Represses gene transcription via multiple mechanisms, including recruitment of histone deacetylase (HDAC) complexes to the target promoter (28, 37). To test this possibility, H1299 cells were treated with the selective HDAC inhibitor TSA 18 h before transfection with the p53 expression vector and E-cadherin-luciferase constructs. TSA increased basal E-cadherin promoter activity (Fig. 8A), suggesting that HDACs participate in the physiological repression of the E-cadherin promoter. Importantly, TSA attenuated p53-mediated repression in all test constructs (Fig. 8A), suggesting that p53 represses the E-cadherin promoter, at least partly, via HDAC-dependent mechanisms.
To correlate the magnitude of histone acetylation in the E-cadherin promoter with the observed transcriptional responses, we performed ChIP analysis in p53-transfected H1299 cells in the presence or absence of TSA treatment. The results revealed that p53 decreased basal E-cadherin promoter H4 acetylation (Fig. 8B), whereas TSA treatment restored promoter-acetylated H4 levels to control levels. Control input DNA samples showed no differences between the treatment groups. In addition, control samples processed with omission of the immunoprecipitation step did not reveal an amplification product (Fig. 8B). These findings are consistent with the functional transcriptional responses whereby TSA restored E-cadherin promoter activity (Fig. 8A).
DISCUSSION
The present study explores the factors underlying aberrant gene regulation and morphogenesis in a gene-environment interaction model of renal dysgenesis. Two lines of independent evidence support the conclusion that p53 mediates repression of E-cadherin gene expression in vivo. First, DNA damage induced by IR is associated with reductions in endogenous E-cadherin mRNA levels in p53+/+ but not p53–/– cells. Second, genetic crosses between B2R–/– and p53+/– resulting in reduced p53 gene dosage restore kidney E-cadherin gene expression. In addition to E-cadherin, the dysplastic kidneys expressed lower levels of other epithelial differentiation genes, including Dlg, HNF-1, and cullin-2. Interestingly, mutations of these genes cause dedifferentiation and cancer (39, 43, 56), which may explain, at least in part, the susceptibility of salt-stressed B2R-null mice to tumorigenesis with aging (23). The mechanisms leading to the downregulation of these tumor suppressors in dysplastic B2R–/– kidneys will be the subject of future investigations.
E-cadherin is a calcium-dependent cell adhesion molecule, which forms a molecular complex with -catenin, plakoglobin, and p120ctn. Through this complex, E-cadherin is connected to the actin cytoskeleton (32, 51, 57). The E-cadherin-catenin adhesion complex is essential for early embryonic development, since E-cadherin-deficient embryos die at the blastocyst stage (29, 44). In addition, E-cadherin has tumor suppressor functions, and mutations affecting its synthesis or transcription occur in many human tumors (4, 7, 10). There is also evidence that E-cadherin functions as a survival factor during terminal epithelial differentiation, since knockdown of E-cadherin expression in the lactating mammary gland induces epithelial cell apoptosis (6). Collectively, the available data suggest that cellular E-cadherin levels must be finely tuned to achieve optimal epithelial sheet adhesion, migration, polarization, and cytoskeleton integrity (40, 55).
p53 Has dual roles as a transcriptional activator or repressor. The consensus p53-response element consists of two copies of the inverted repeats (RRRCA/TT/AGYYY), separated by 0–13 bp (8, 17, 27, 54). The mechanisms of p53-mediated transcriptional repression are far less well understood than those of activation. Recruitment of HDACs, displacement of activators, or interactions with the basal transcription machinery are potential mechanisms of p53-mediated repression (18, 26, 30, 33, 34). The results of this study suggest that direct binding of p53 to the E-cadherin promoter is required, although not sufficient for p53-mediated repression since 1) a mutant p53 that cannot bind DNA did not repress the E-cadherin promoter and 2) mutation of the p53-binding site attenuated but did not eliminate p53-induced repression. It is possible that p53 represses E-cadherin transcription partly by interacting with and squelching transcriptional activators (18). Another mechanism by which p53 could influence E-cadherin expression is through binding with Smad2 (11). Smad2 represses E-cadherin expression, leading to epithelial-mesenchymal transition (31).
Acetylation of histone NH2 termini by histone acetylases (e.g., CBP/p300) alters chromatin conformation, allowing transcriptional regulators better access to DNA-regulatory elements (2). The balance between histone acetylase and HDAC activity is an important determinant of gene transcription. Previous studies have demonstrated that p53 represses certain genes via recruitment of HDAC-containing repressor complexes (36). Experiments that utilized TSA to inhibit endogenous HDAC activity revealed increased baseline E-cadherin promoter activity and attenuation of p53-mediated repression. In addition, ChIP assays revealed that TSA treatment of p53-transfected cells restores histone H4 acetylation in the E-cadherin core promoter region. One of our future goals is to adapt the ChIP technique to embryonic tissues to map occupancy of p53-HDAC complexes on the endogenous E-cadherin gene. Reestablishing expression of E-cadherin (and other differentiation genes) by HDAC inhibitors may offer a therapeutic strategy in experimental renal dysgenesis, similar to what has been described in cancer (35, 52).
The mechanisms leading to activated p53 in this model of renal dysgenesis are not completely clear. Our proteomic screen has shown that although Chk1 is upregulated 19.5- fold, P-Ser20-p53 is only increased 2.5-fold in dysplastic B2R–/– kidneys. The difference may represent differential antibody sensitivity or, more likely, the fact that full p53 stabilization requires additional posttranslational modifications including NH2-terminal phosphoprylation and COOH-terminal acetylation (38). IR-induced Ser20 phosphorylation is mediated via Chk1 activation (48). At first glance, these data suggest that the pathogenesis of renal dysgenesis mimics that of genotoxic stress. However, DNA damage-induced Chk1 activation results from phosphorylation by upstream kinases (e.g., ATM/ATR) rather than increased protein levels, as is observed in dysplastic kidneys. Moreover, p53 is known to repress transcription of the human Chk1 gene (13). These findings suggest that DNA damage as well as other pathways leading to the activation of p53 may be involved in the susceptibility of B2R-null mice to renal dysgenesis. It is unknown yet whether maternal high salt crosses the placenta to reach the fetal circulation. Studies conducted in cultured inner medullary collecting duct cells have shown that high salt induces DNA strand breaks and activates p53 (15).
The full biological significance of p53-mediated repression of E-cadherin gene expression is likely to be context dependent. Since E-cadherin function is required for cell survival (3, 6, 14), it is conceivable that p53-mediated repression of E-cadherin is a mechanism to promote cell death (19). Preliminary studies in our laboratory indicate that B2R-null dysplastic kidneys exhibit widespread apoptosis and that rescue of renal dysgenesis by reduction of p53 gene dosage (shown in the current study) is accompanied by restriction of cell death (Fan H and El-Dahr SS, unpublished observations). In addition, downregulation of E-cadherin by p53 can promote epithelial-mesenchymal transition (49), which may explain expansion of the mesenchymal stroma typically observed in dysplastic kidneys.
GRANTS
These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-56264 and DK-62250, the Tulane Renal and Hypertension Center of Excellence, and the American Heart Association, Southeast Affiliate.
ACKNOWLEDGMENTS
We thank Drs. Eric Fearon and Bert Vogelstein for providing the E-cadherin promoter constructs and HCT116 cells, respectively.
FOOTNOTES
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.
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