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Molecular Cancer Research 4:759-768 (2006)
© 2006 American Association for Cancer Research

Signaling and Regulation

p63 Overexpression Induces the Expression of Sonic Hedgehog

Tina M. Caserta1,2, Ramakrishna Kommagani1, Ziqiang Yuan3, David J. Robbins3, Carol A. Mercer1 and Madhavi P. Kadakia1,2

1 Department of Biochemistry and Molecular Biology, 2 Center for Genomics Research, Wright State University, Dayton, Ohio; and 3 Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire

Requests for reprints: Madhavi P. Kadakia, Department of Biochemistry and Molecular Biology, Wright State University, Dayton, OH 45435. Phone: 937-775-2339; Fax: 937-775-3730. E-mail: madhavi.kadakia{at}wright.edu


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
p63 and p73 are members of the p53 protein family and have been shown to play an important role in cell death, development, and tumorigenesis. In particular, p63 has been shown to be involved in the maintenance of epidermal stem cells and in the stratification of the epidermis. Sonic Hedgehog (Shh) is a morphogen that has also been implicated to play a role in epithelial stem cell proliferation and in the development of organs. Recently, Shh has also been shown to play an important role in the progression of a variety of cancers. In this report, we show that p63 and p73 but not p53 overexpression induces Shh expression. In particular, p63{gamma} and p63ß (both TA and {Delta}N isoforms) and TAp73ß isoform induce Shh. Expression of Shh was found to be significantly reduced in mouse embryo fibroblasts obtained from p63–/– mice. The naturally occurring p63 mutant TAp63{gamma}(R279H) and the tumor suppressor protein p14ARF inhibited the TAp63{gamma}-mediated transactivation of Shh. The region –228 to –102 bp of Shh promoter was found to be responsive to TAp63{gamma}-induced transactivation and TAp63{gamma} binds to regions within the Shh promoter in vivo. The results presented in this study implicate p63 in the regulation of the Shh signaling pathway. (Mol Cancer Res 2006;4(10):759–68)


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
p63 is a homologue of the tumor suppressor protein p53 and is known to play a role in tumorigenesis, maintenance of epithelial stem cells, and development (1-3). p63 has a complex genomic organization that results from alternate promoter usage and alternative splicing. The p63 gene has two major promoters that are used to generate transcripts encoding proteins with or without the transactivation domain which are referred to as the TA and {Delta}N p63 isoforms, respectively (1). Additionally, the differential splicing of the p63 gene results in p63 isoforms with different COOH termini termed {alpha}, ß, and {gamma}. The {alpha} and {gamma} isoforms of p63 are the most widely studied, whereas little is known about the ß isoforms. When compared with TAp63{alpha}, TAp63{gamma} is a more potent transactivator (1, 4). Unlike p53 knockout mice that exhibit a high incidence of spontaneous tumors (5), p63 knockout mice display severe developmental abnormalities including limb truncations and craniofacial abnormalities (2, 3).

p63 has been implicated in the maintenance of epidermal stem cells and in the stratification of the epidermis. TAp63 isoforms are required for the initiation of an epithelial stratification program, whereas {Delta}Np63 isoforms are required for the maintenance of the mature epidermis, demonstrating that p63 is essential not only for the commitment of ectoderm to stratified epithelia but also for the proliferative potential of epithelial stem cells (2, 3, 6). TAp63{gamma} has also been shown to activate the expression of Jagged 1 and Jagged 2, which are involved in Notch signaling, whereas Notch 1 activation has also been shown to suppress {Delta}Np63{alpha} expression, demonstrating a cross-talk between these pathways (7, 8). Additional studies have shown that the biological effects of p63 in development and tumor suppression is exerted through the regulation of multiple signaling pathways (9-17). p73, another member of the p53 family, was also discovered based on structural similarities to p53 (18). Like p63, p73 also exists as TA and {Delta}N isoforms based on promoter usage and alternate COOH-terminal splicing. p73 knockout mice exhibit defects in neuronal development (19, 20).

Sonic Hedgehog (Shh) is a morphogen that is essential for the development of the brain, limbs, prostate, hair follicles, and teeth (21-23). The Shh signaling pathway controls the proliferation of precursor cells with stem cell properties (24). Shh is involved in ductal morphogenesis, regeneration of prostate epithelium, and prostate cancer progression (23). Although Shh is an important regulator of development, the regulatory mechanisms responsible for Shh expression remain unclear.

Both p63 and Shh have been shown to play a role in the proliferation of epithelial cells. Furthermore, mice lacking p63 exhibit severe developmental defects such as partial or total limb truncations, abnormal skin, lack of the prostate, and absence of hair follicles, teeth and/or mammary glands, all of which require Shh for proper development (2, 3). In this study, we show that TAp63ß, TAp63{gamma}, {Delta}Np63{gamma}, {Delta}Np63ß, and TAp73ß activate Shh gene expression. TAp63{gamma} was found to bind to the Shh promoter in vivo. Shh expression was significantly reduced in mouse embryo fibroblasts (MEF) from p63–/– mice, suggesting that the developmental defects observed in p63–/– knockout mice might occur as a result of the Shh signaling pathway deregulation.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
p63ß, p63{gamma}, and p73ß Induce the Expression of Shh
To investigate the role of p63 isoforms in regulating the expression of Shh, we first tested the ability of TAp63{alpha}, TAp63ß, TAp63{gamma}, {Delta}Np63{alpha}, {Delta}Np63{gamma}, and {Delta}Np63ß to induce Shh gene expression. In addition, the effects of TAp73ß, {Delta}Np73ß, and p53 on Shh expression were also tested. H1299 cells (devoid of endogenous p53 and lacking detectable p63 protein) were transfected with p63 isoforms, TAp73ß, {Delta}Np73ß, p53, or empty vector backbone and the relative Shh transcript levels were assessed using TaqMan reverse transcriptase-PCR. As shown in Fig. 1A , cells transfected with both TA and {Delta}N isoforms of p63ß and p63{gamma} showed a significant increase in the expression of Shh. In contrast, cells transfected with TAp63{alpha}, {Delta}Np63{alpha}, and p53 exhibited a modest increase in Shh expression. Our observation that TAp63{alpha} is less effective in inducing Shh expression relative to TAp63{gamma} is consistent with previous studies in which TAp63{gamma} is shown to be a more effective transactivator compared with TAp63{alpha} (1, 25, 26). p73 is another member of the p53 family and is closely related to p63. Due to the high degree of sequence similarity between the DNA-binding domains of p63 and p73, it is likely that p73 might also be able to activate the expression of Shh. H1299 cells transfected with TAp73ß but not {Delta}Np73ß showed a significant increase in the Shh transcript levels (Fig. 1A). The expression of p21, a known target of p53 family members, was also examined in cells transfected with p53, p63, and p73. As expected, the TAp63 isoforms, TAp73ß, and p53 significantly up-regulated the expression of p21.


Figure 1
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  		FIGURE 1. p63 and p73 up-regulates Shh expression and Shh reporter activity. H1299 cells were transfected with the indicated expression plasmids. A. Shh and p21 transcript levels detected using TaqMan reverse transcriptase-PCR. Y-axis, the fold change in transcript levels calculated using the Pfaffl method when compared with cells transfected with empty vector control. B. Immunoblot analysis of cell extracts harvested at 24 hours post-transfection was resolved on a 12% SDS-PAGE and probed using anti-p63, anti-p73, anti-p53, anti-Shh, anti-p21, and anti-ß actin. C and D. H1299 cells were transfected with pShhFL-Luc reporter plasmid along with the indicated expression plasmids. Y-axis, relative luciferase units normalized to protein levels (RLU/protein) or transfection efficiency (RLU/ß-gal). pGL3 basic vector (lacking the Shh promoter region) cotransfected with p63 or p73 showed no significant change in the reporter luciferase counts (data not shown).

 
The increase in Shh transcript levels in cells transfected with both TA and {Delta}N isoforms of p63{gamma} and p63ß as well as TAp73ß resulted in a corresponding increase in Shh protein levels. Shh protein levels were also found to be higher in cells transfected with TAp63{gamma} and TAp63ß than in {Delta}Np63{gamma}-, {Delta}Np63ß-, or TAp73ß-transfected cells, whereas Shh protein was barely detectable in cells transfected with TAp63{alpha}, {Delta}Np63{alpha}, or p53 (Fig. 1B). Shh protein is expressed as a 45 kDa precursor that undergoes intramolecular cleavage to generate an amino terminal polypeptide ShhN and a carboxyl-terminal polypeptide ShhC (27). During this cleavage, cholesterol is covalently attached to the last amino acid of the amino terminal polypeptide of Shh to create ShhNp (p stands for processed). The cholesterol-modified form, ShhNp, migrates faster than ShhN. In order to verify which of the two forms of Shh accumulates in cells overexpressing p63, we compared the electrophoretic mobility of the Shh protein in p63-overexpressing cells to cell extracts containing either the ShhN or ShhNp by immunoblot analysis (Fig. 1B). Based on the electrophoretic mobility of the Shh protein, ShhNp (the processed form) accumulates in H1299 cells upon p63 and p73 overexpression. As expected, immunoblot analysis of p21 revealed that all TAp63 isoforms, TAp73ß, and p53, significantly up-regulate p21 protein levels, consistent with the results obtained using TaqMan reverse transcriptase-PCR (Fig. 1A and B).

A Shh promoter-luciferase fusion was constructed and was used to validate that the activation of Shh by p63 is due to an increase in transcriptional activity and not a change in mRNA stability. The 703 bp promoter region of Shh was fused upstream of the luciferase reporter gene in the pGL3 basic plasmid (28). The transactivation of the Shh promoter-luciferase fusion (pShhFL) was investigated by cotransfecting Shh reporter, pShhFL along with increasing doses of TAp63{gamma}, TAp63{alpha}, and p53 into H1299 cells. A dose-dependent increase in pShhFL reporter activity was observed with increasing concentrations of TAp63{gamma}, whereas TAp63{alpha} and p53 showed only a modest change in the reporter activity (Fig. 1C). The reporter activity in the transfected cells was assessed using two different methods, normalization to protein levels or to transfection efficiency, and the overall trend in reporter activity was found to be similar. To assess whether TAp73ß also up-regulates Shh reporter, H1299 cells transfected with TAp63{gamma}, TAp63{alpha}, TAp73ß, p53, and empty vector controls. The results showed a significant increase in the Shh reporter activity in cells transfected with TAp63{gamma} followed by TAp73ß (Fig. 1D). We also observed that transactivation of Shh expression by p63 was not limited to the H1299 cell line because the HeLa and the PC3 prostate cancer cell lines also showed similar results (data not shown).

Taken together, these results show that cells transfected with TAp63ß, TAp63{gamma}, {Delta}Np63ß, and {Delta}Np63{gamma} isoforms induce the expression of Shh. Although TAp73ß is most closely related to TAp63{gamma}, induction of Shh expression by TAp73ß was comparatively lower. Although both the TAp63ß and TAp63{gamma} isoforms of p63 induces Shh expression, we chose to concentrate our studies on TAp63{gamma} for the remaining set of experiments described in this article.

p63–/– MEFs Exhibit Reduced Shh Expression
Shh has been shown to be essential for the development of brain, limbs, prostate, teeth, and hair follicles (21-23). If p63 is indeed involved in the expression of Shh, then a reduction in the levels of Shh would be expected in cells lacking p63. We evaluated the expression of Shh in primary MEFs obtained from p63–/– and p63+/+ mice. As shown in Fig. 2A , Shh transcript levels were significantly reduced in the p63–/– MEFs when compared with p63+/+ cells. In contrast, the levels of Id2 (a negative regulator of basic helix-loop-helix transcription factors and involved in cellular differentiation and proliferation), which is not a p63 target, showed only a modest change in transcript levels in p63–/– mice. Interestingly, immunoblot analysis of p63–/– MEF cell extract shows the presence of low levels of p63. This suggests that the p63–/– MEF may not exhibit a complete lack of p63 expression when compared with the wild-type MEFs (Fig. 2B). Nonetheless, the reduced transcript levels of Shh in p63–/– MEFs is commen-surate with the reduced levels of p63.


Figure 2
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  		FIGURE 2. Shh expression requires p63. A. Shh, p63, and Id2 transcript levels in primary MEFs obtained from either p63+/+ or p63–/– mice determined using TaqMan-based reverse transcriptase-PCR. Y-axis, the levels of Shh, p63, and Id2 expression in p63–/– MEFS when compared with wild-type MEFs (p63+/+). B. Immunoblot analysis of E1A-transformed p63+/+ or p63–/– MEFs using anti-p63. C. H1299 cells were transfected with pShhFL-Luc reporter plasmid alone or cotransfected along with vector control, TAp63{gamma}, or TAp63{gamma}(R279H). Equivalent amounts of cell extracts were resolved by SDS-PAGE and immunoblotted with anti-p63 antibody or anti-Shh antibody. D. H1299 cells were transfected with pShhFL-Luc reporter plasmid alone or with GST-TAp63{gamma} alone or with increasing concentrations of HA-tagged TAp63{gamma}(R279H) as indicated. Bottom, overexpression of GST-tagged TAp63{gamma} and HA-tagged TAp63{gamma}(R279H) was confirmed by immunoblot analysis using anti-p63 antibody. Immunoblotting with anti-ß-actin serves as a loading control.

 
TAp63{gamma} (R279H) Mutant Fails to Transactivate Shh Expression
Single amino acid mutations in p63, such as the R279H missense mutation, have been frequently detected in patients with ectrodactylyl, ectodermal dysplasia, and facial cleft–like syndrome (29). Because the R279H missense mutation localizes to the DNA-binding domain and does not activate p63 target genes, we tested whether this mutant affected Shh expression. TAp63{gamma} or the TAp63{gamma}(R279H) mutant, along with the pShhFL reporter, were cotransfected into H1299 cells. Cells transfected with TAp63{gamma}(R279H) failed to exhibit an increase in the expression of Shh reporter activity (Fig. 2C). Additionally, Shh protein levels were not up-regulated in cells transfected with the p63{gamma}(R279H) mutant (Fig. 2C, bottom). Although TAp63{gamma}(R279H) was unable to induce Shh gene expression, the potential for the mutant to heterotetramerize with the wild-type p63 suggested that it might affect Shh gene expression. As shown in Fig. 2C, TAp63{gamma}-induced transactivation of the Shh reporter was significantly inhibited by the TAp63{gamma}(R279H) mutant in a dose-dependent manner. These results indicate that the DNA binding domain of TAp63{gamma} is required for the activation of Shh expression, and that TAp63{gamma}(R279H) can act in a dominant-negative manner to inhibit TAp63{gamma}-induced transactivation of Shh, suggesting that heterotetramers formed between p63 and p63 mutant forms might interfere with the function of p63 involved in transactivating the expression of its target genes.

The Tumor Suppressor p14ARF Inhibits TAp63{gamma}-Induced Transactivation of Shh
p14ARF is a tumor suppressor protein that activates p53 function by stabilizing p53 and blocking Mdm2-mediated inhibition of p53 degradation (30). p14ARF was also shown to negatively regulate the transcriptional activity of p63 (31). We have recently shown that p14ARF inhibits TAp63{gamma}-mediated induction of vitamin D receptor (13). Here, we examined whether p14ARF affected TAp63{gamma}-mediated induction of Shh expression. We observed that p14ARF was able to inhibit TAp63{gamma}-mediated transactivation of pShhFL reporter activity (Fig. 3B ). As a positive control, p14ARF was able to inhibit the p63{gamma}-mediated transactivation of PG13-Luc, a reporter construct containing 13 copies of the p53 binding consensus sequence upstream of a luciferase gene which is also recognized by TAp63{gamma} (Fig. 3A). We showed that coexpression of TAp63{gamma} and p14ARF led to a reduction in endogenous Shh transcript levels and a loss of Shh protein when compared with cells expressing TAp63{gamma} alone (Fig. 3C). These results further confirm the specificity of TAp63{gamma} in the transactivation of Shh and the ability of p14ARF to inhibit the transactivation of another TAp63{gamma} target gene.


Figure 3
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  		FIGURE 3. p14ARF inhibits p63{gamma}-mediated transactivation of Shh. H1299 cells were transfected with (A) PG-13 Luc or (B) pShhFL-Luc reporter alone or cotransfected with TAp63{gamma} and p14ARF expression plasmids as indicated. Overexpression of TAp63{gamma} and p14ARF was confirmed by immunoblot analysis using anti-p63 and anti-ARF antibodies, respectively. C. Shh transcript levels in H1299 cells transfected with TAp63{gamma} and p14ARF expression plasmids using TaqMan reverse transcriptase-PCR. Bottom, equivalent amounts of whole cell extracts were resolved by SDS-PAGE and immunoblotted with anti-Shh, anti-p63, and anti-ARF. Immunoblotting with anti ß-actin serves as a loading control.

 
Transactivation of Shh by TAp63{gamma} Does Not Require De novo Protein Synthesis
In order to elucidate the mechanism by which TAp63{gamma} transactivates Shh expression, we created a TAp63{gamma}-inducible expression system that can be conditionally activated by estrogen hormones. The ligand-binding domain of the human estrogen receptor (ER) was fused to TAp63{gamma}. In the absence of the ligand 4-hydroxy-tamoxifen (OHT), the ER-TAp63{gamma} fusion protein is bound to the heat shock protein Hsp90 and remains localized in the cytoplasm. Treatment with the ER ligand (OHT) results in the dissociation of Hsp90, nuclear translocation of the ER-TAp63{gamma}, and transactivation of target genes (32). We first characterized the ER-TAp63{gamma} fusion protein using immunofluorescence and transactivation assays. Immunofluorescence staining of H1299 cells transfected with ER-TAp63{gamma} confirmed OHT-dependent nuclear translocation of ER-TAp63{gamma} (data not shown). To assess the biological activity of the ER-TAp63{gamma} fusion protein, the transactivation of pShhFL-Luc reporter or PG13-Luc reporter in H1299 with or without the ligand OHT was determined. Cells transfected with ER-TAp63{gamma} exhibit an increase in pShhFL and PG13-luc reporter activity only upon OHT treatment (Fig. 4A ). Although ER-TAp63{gamma} protein expression was independent of OHT treatment, the transactivation of the reporters by ER-TAp63{gamma} occurs only when cells were treated with OHT (Fig. 4A, lower panel). The increase in Shh reporter activity in ER-TAp63{gamma}-transfected cells cultured in the presence of OHT is inhibited by TAp63{gamma}(R279H) mutant (Fig. 4B). The inducible expression of Shh by ER-TAp63{gamma} in H1299 cells was also confirmed by reverse transcriptase-PCR (Fig. 4C). As expected, ER-TAp63{gamma}-mediated induction of Shh expression was inhibited by TAp63{gamma}(R279H) mutant (Fig. 4C). H1299 cells transfected with empty vector control in the presence or absence of OHT did not affect Shh transcript levels (data not shown).


Figure 4
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  		FIGURE 4. Transactivation of Shh by p63{gamma} does not require de novo protein synthesis. A. The activity of pShhFL-Luc or PG-13-Luc reporter construct in H1299 cells cotransfected with ER-TAp63{gamma} and cultured in the presence or absence of tamoxifen (OHT). Bottom, overexpression of ER-TAp63{gamma} was confirmed by immunoblot using anti-p63 antibody. B. H1299 cells transfected with pShhFL-Luc reporter plasmid along with ER-TAp63{gamma} and GST-tagged TAp63{gamma}(R279H) expression plasmids. Y-axis, relative luciferase units normalized to protein levels (RLU/protein). In the lower panel, the overexpression of ER-TAp63{gamma} and GST-TAp63{gamma}(R279H) was confirmed by immunoblot analysis using anti-ER and anti-GST antibodies, respectively. C. Shh transcript levels in H1299 cells transfected with ER-TAp63{gamma} alone or along with GST-TAp63{gamma}(R279H) expression plasmid, as indicated, were pretreated with translational inhibitors cycloheximide or anisomycin for 1 hour followed by treatment with OHT for 6 hours as indicated. Y-axis, fold change in Shh transcript level detected by TaqMan reverse transcriptase-PCR relative to cells transfected with ER-TAp63{gamma} and cultured in absence of OHT (lane 1).

 
A distinguishing feature of the ER system is that the activation of the fusion protein by the ligand can occur in the absence of de novo protein synthesis. Activation of ER-TAp63{gamma} by OHT while protein synthesis is inhibited can be used to differentiate between direct and indirect targets of TAp63{gamma} (33). To address this, H1299 cells transfected with ER-TAp63{gamma} were pretreated with the protein synthesis inhibitors anisomycin or cycloheximide for 1 hour and subsequently cultured in the presence or absence of OHT. At 6 to 7 hours post-OHT treatment, RNA was extracted from the cells and Shh mRNA levels were quantitated using TaqMan reverse transcriptase-PCR. As shown in Fig. 4C, activation of Shh by TAp63{gamma} persisted even in the presence of protein synthesis inhibitors. Cotransfection of TAp63{gamma}(R279H) inhibited the up-regulation of Shh mRNA by ER-TAp63{gamma}, hence, confirming that this is most likely a TAp63{gamma}-specific effect (Fig. 4C). Taken together, the results show that transactivation of Shh does not require de novo protein synthesis; thus, ruling out the possibility of an autocrine mechanism and suggesting that TAp63{gamma} is likely inducing the expression of Shh directly.

Identification of the Minimal Promoter Region of Shh Responsive to TAp63{gamma}
A series of 5' and 3' Shh promoter deletions were constructed in order to define the minimal region within the Shh promoter required for TAp63{gamma}-induced transactivation. H1299 cells were transfected with the promoter deletion reporter plasmids alone or along with the TAp63{gamma} expression plasmid. Based on the promoter deletion as shown in Fig. 5A and B , it seems that region between –228 and –1 bp from the Shh promoter is required for TAp63{gamma}-dependent transactivation (Fig. 5A). A significant reduction in the transactivation of Shh by TAp63{gamma} was observed when the region between –228 and –102 bp is eliminated (Fig. 5B). Taken together, these results implicate the –228 to –102 bp region of the Shh promoter as the minimal region required for TAp63{gamma}-induced transactivation. The possibility that the –228 to –102 region of the Shh promoter contained the elements required for TAp63{gamma}-induced transactivation was further evaluated. The –228 to –102 region of the Shh reporter was cloned into the pGL3 basic vector and transfected into H1299 cells. As shown in Fig. 5C, the region –228 to –102 of the Shh promoter was responsive to TAp63{gamma}-induced transactivation but not to TAp63{alpha} or p53. This supports that the region –228 to –102 of the Shh promoter region contains element(s) to confer both transactivation potential and specificity. Studies examining the promoter region of p63-responsive genes has resulted in the identification of responsive elements that are specifically activated by p63 (15, 34). The p63-responsive element seems to differ from the canonical (RRRCWWGYYY)-responsive element of p53 in the CWWG core binding element as well as in the flanking RRR and YYY stretches (34). The nucleotide G in the fifth position, instead of a W, within the core along with a number of mismatches in the flanking RRR and YYY sequence was found to distinguish the p63-responsive elements from that of p53 (34). In addition, the core sequence CAAG has been shown to preferentially bind p63. Sequence analysis of the Shh promoter region between –228 and –102 uncovered five potential p63 binding sites. The core CWWG element with the flanking RRR and YYY sequences as well as mismatches in the flanking regions are shown in Fig. 5D. This observation further supports the data obtained by our deletion analysis which indicates the region between –228 and –102 to be important for TAp63{gamma}-induced transactivation.


Figure 5
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  		FIGURE 5. p63 responsive region in the Shh promoter and binding of TAp63{gamma} to the Shh promoter in vivo. H1299 cells were transfected with the indicated (A) 5' deletions and (B) 3' deletions of the Shh promoter and TAp63{gamma} expression plasmid. The numbers indicate the DNA fragment spanning the Shh promoter region. Y-axis, relative luciferase units normalized to protein levels (RLU/protein). In the lower panels, equivalent amount of cell extracts based on protein concentrations were resolved by SDS-PAGE and immunoblotted with anti-p63 to confirm overexpression of p63. C. The Shh-Luc reporter plasmid containing the promoter region spanning –228 to –102 was transfected into H1299 cotransfected with vector control, TAp63{gamma}, TAp63{alpha} or p53 expression plasmid. Y-axis, relative luciferase units normalized to protein levels (RLU/protein). Immunoblot analysis using anti-p63 and anti-p53 (bottom). D. A schematic representation of the potential p63-binding nucleotide sequences within the Shh promoter region –228 to –102. Mismatches within the putative p63 binding elements (*); the putative TATA box (underlined). E. Schematic of the Shh promoter region and the five overlapping regions amplified by PCR. F. Agarose gel electrophoresis of the PCR products from the ChIP assay using 1% input sonicated chromatin prior to immunoprecipitation, chromatin immunoprecipitated with anti-p63, and normal rabbit IgG from TAp63{gamma}-transfected cells. G. Agarose gel electrophoresis of the PCR products from the ChIP assay for sites 1, 3, and 5 using 1% input sonicated chromatin prior to immunoprecipitation (lane 1), chromatin immunoprecipitated with anti-p53 (lane 2), and normal rabbit IgG (lane 3) from p53-transfected cells. 14-3-3{sigma} was used as a positive control because both p53 and p63 binds to its promoter region.

 
p63{gamma} Binds to Regions Within the Shh Promoter
To unequivocally show that Shh is a direct target of TAp63{gamma}, we examined the ability of p63 to bind to the Shh promoter region in vivo using chromatin immunoprecipitation (ChIP) assays. H1299 cells transfected with TAp63{gamma} or p53 expression plasmids were cross-linked by exposure to 1% formaldehyde as described in Materials and Methods. After cross-linking, equal amounts of precleared sonicated chromatin were immunoprecipitated with p63-specific or p53-specific antibodies and the DNA to which p63 or p53 protein bound was purified. The immunoprecipitated DNA complexes were amplified by PCR using primers specific to overlapping regions of Shh promoter region (Fig. 5E). To control for nonspecific binding during immunoprecipitation, cross-linked lysates were immunoprecipitated with normal rabbit serum, which does not cross-react with either p63 or p53. As shown in Fig. 5F, TAp63{gamma} bound to the Shh promoter site 1 (–702 to –467) and site 5 (–228 to –1). No binding of TAp63{gamma} was observed at sites 2 to 4 of the Shh promoter. p53 was unable to bind to sites 1, 3, and 5 of the Shh promoter (Fig. 5G). Both p53 and p63 were able to bind to the promoter region of a previously known target gene 14-3-3{sigma}. Therefore, the binding regions within sites 1 and 5 seem to be specific to p63. Based on these ChIP experiments, we show that p63 binds to the Shh promoter and that the transactivation of Shh expression by TAp63{gamma} occurs as a result of TAp63{gamma} binding to the Shh promoter. The binding of p63 to site 1 (–702 to –467) can be due to the potential p63 binding sites (–703 GTGCTTGATG –691 and –686 AAGCTTGCGG –677). Although the region –703 to –467 of the Shh promoter binds to p63, transactivation data using reporter assays shows that this region is not activated by TAp63{gamma} (Fig. 5B). This suggests that binding between a transcriptional factor and responsive element, although necessary, is not sufficient for transactivation. More importantly, binding of p63 to site 5 (–228 to –1) support the promoter deletion data as well as promoter sequence analysis, which suggests that several candidate sites exists within the Shh promoter for p63 binding.


   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
The identification of p63 (specific) targets is critical in order to fully understand how p63 functions. Although it is becoming clear that p63 plays a role in development as well as in tumorigenesis, studies identifying the target genes preferentially regulated by p63 are currently under way. The overlapping roles of signaling pathways in cellular proliferation and cancer progression suggests that transcription factors such as p63 may be playing a critical role modulating the activities of these pathways. The Shh pathway has been shown to play an important role in carcinogenesis and development. Given the similarities in the role of p63 and Shh in both tumorigenesis and developmental disorders, we hypothesized that p63 might be involved in regulating Shh expression. The results presented in this study show that both TA and {Delta}N isoforms of p63{gamma} and p63ß induces Shh expression. It should be noted that although the DNA binding domain is conserved between TAp63{gamma} and TAp63{alpha}, modest Shh induction by TAp63{alpha} can be due to the carboxyl-terminal transactivation inhibitory domain present only in the TAp63{alpha} isoform (Fig. 1; ref. 35). Although the {Delta}N isoforms of p63 lack the transactivation domain, they do retain the capacity to transactivate certain genes such as vascular epithelial growth factor and heat shock protein 70 (16, 36-38). Our study further confirms that the {Delta}Np63{gamma} and {Delta}Np63ß isoforms could also induce Shh expression.

p63 has a high degree of sequence homology to p73, suggesting that they may target similar genes. p73 has been shown to exhibit defects in neuronal development, but more recently, p63{gamma} has also been implicated in neuronal development (39). Shh has also been shown to play a major role in neuronal development, therefore, regulation of Shh by TAp73ß most closely related to TAp63{gamma} was also investigated. We showed that p73 also induces Shh expression. These results show that p63 and p73, the two most closely related members of the p53 family, may play a role in development through the regulation of common target genes such as Shh.

The inability of the TAp63{gamma}(R279H) missense mutation to induce Shh expression was not due to a lack of steady state protein levels because the TAp63{gamma}(R279H) mutant seems to be more stable than wild-type TAp63{gamma} (Fig. 2). It is plausible that mutations within p63, such as TAp63{gamma}(R279H), may be responsible for the developmental disorders as a result of a defect in the regulation of the Shh signaling pathway. The regulation of Shh by p63 is considered specific because both TAp63{gamma}(R279H) mutant, as well as p14ARF, a negative regulator of p63 function, inhibited p63{gamma}-mediated transactivation of Shh reporter activity.

The region within the Shh promoter responsive to TAp63{gamma}-induced transactivation was shown to be located between nucleotides –228 and –102 relative to the transcriptional start site of Shh and contains several potential p63 binding sites. It is clear that the regulation of Shh promoter by p63 is complex and will require additional studies to carefully define regulatory elements and provide clues to a DNA consensus element necessary for p63 binding. Interestingly, p53 does not transactivate Shh expression. When the Shh promoter sequence was analyzed for p53 DNA consensus binding sequences using the PATCH program (http://www.gene-regulation.com/cgi-bin/pub/programs/patch/bin/patch.cgi), a potential p53 half-binding site within site 3 of the Shh promoter was uncovered which exhibited limited similarity to the canonical p53 binding site. However, p53 was unable to bind to site 3, as seen by ChIP analysis (Fig. 5G). Recently several studies have examined the potential p63 DNA binding consensus sites within p63-responsive genes (14, 15, 17). In these studies, p63 was shown to preferentially bind to DNA elements that are distinct from the p53 DNA binding element.

Therefore, the results presented in our study are consistent with previous observations which indicate that p63 can regulate the expression of specific target genes such as those involved in skin, limb, and craniofacial development via distinct p63-specific response elements. In conclusion, we report that Shh is induced by p63 and point to a potential role for p63 in the regulation of the Shh signaling pathway.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Plasmids
Carboxyl-terminal c-myc/his tagged TAp63{gamma} and TAp63{alpha} expression plasmids were constructed as described earlier (40). A CMV promoter-driven plasmid was used to construct GST-TAp63{gamma} and HA-tagged TAp63{gamma} expression plasmids. GST- and HA-tagged TAp63{gamma}(R279H) mutants were created using PCR-based site-directed mutagenesis method using sense (5'-GGAGGGATGAACCACCGTCCAATTTTAATC-3') and antisense (5'-GATTAAAATTGACGGTGGTTCATCCCTCC-3') primers. A reporter plasmid expressing luciferase under the control of the Shh promoter was constructed using BAC clone RP11-6903 containing the region upstream of the Shh open reading frame –703 to –1 (BACPAC Resources, Oakland, CA; ref. 28). The Shh promoter region (–703 to –1) was amplified by PCR using sense (5'-GAGCTCTCTGTGCTTGATGACTGAAGC-3') and antisense (5'-CTCGAGCTCGC CCATGGAACTGATGAC-3') primers and cloned into the promoterless pGL3 Basic vector (Promega, Madison, WI) at the SacI and XhoI sites. Serial promoter deletions of the Shh promoter containing regions –467 to –1 (sense 5'-GAGCTCCAGCAGCAACAGAAAAAAAA-3'), –342 to –1 (5' sense 5'-GAGCTCCACAAGCTCTCCAGGCTTGC-3'), –228 to –1 (sense 5'-GAGCTCCCTGTCTGGGTGGGGAGCGG-3'), and –102 to –1 (sense 5'-GAGCTCAGG GAAAGCGCAAGAGAGAG-3') were constructed by amplifying the desired region by PCR from the BAC clone RP11-6903 using specific sense primers and antisense primer similar to the full-length Shh promoter. Another set of serial promoter deletions of the Shh promoter containing regions –703 to –467 (antisense 5'-CTCGAGTTGCTTTATTTTCACCTGAAG-3;), –703 to –342 (antisense 5'-CTCGAGAGAGAGGCTGCCTTTAGCACT-3'), –703 to –228 (antisense 5'-CTCGAGGGCTGGAATGGCAGGCTGCCG-3'), and –703 to –102 (antisense 5'-CTCGAGTCCTCGCTCCGGCTCGCCCGC-3') using specific antisense primers and the sense primer similar to the full-length promoter. The region –228 to –102 was cloned using sense (5'-CGGGGTACCCAGCCCCTGTCTGGGTGGGGAGCG-3') and antisense (5'-CCCAAGCTTTTCCTCGCTCCGGCTCGCCCGCTCGCT-3') primers. The PCR fragments were also cloned into the promoterless pGL3 Basic vector plasmid at the SacI and XhoI sites. Expression plasmids encoding ShhN and ShhNp used for positive controls were previously described (41). The ER-TAp63{gamma} expression plasmid was constructed by cloning the TAp63{gamma} cDNA into a CMV promoter-driven expression vector such that the ligand-binding domain of the ER was fused to the amino termini of p63{gamma} (32). The PG13-Luc reporter, p14ARF and p53 expression plasmids were kindly provided by Dr. Steven Berberich (Wright State University, Dayton, OH). The expression plasmids encoding the TAp63{gamma}, {Delta}Np63{gamma}, and {Delta}Np63{alpha} were kindly provided by Dr. Frank McKeon (Harvard Medical School, Boston, MA) and the expression plasmids encoding TAp73ß, {Delta}Np73ß, TAp63ß, and {Delta}Np63ß were kindly provided by Dr. Xinbin Chen (University of Alabama, Birmingham, AL).

Cell Lines and Transfection
H1299 cells, a non–small lung carcinoma cell line devoid of p53, were maintained in DMEM supplemented with 10% fetal bovine serum and 50 units/mL of penicillin and 50 µg/mL of streptomycin. Primary MEFs obtained from wild-type or p63–/– mice and E1A-transformed MEFs obtained from wild-type or p63–/– mice were obtained from Dr. Elsa Flores (University of Texas M.D. Anderson Cancer Center, Houston, TX). H1299 cells were transfected with the desired plasmids using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and harvested at 24 hours post-transfection as described previously (40).

Protein Isolation and Immunoblot Analysis
For Shh expression studies, at 24 hours post-transfection, medium was removed and plates were kept on ice and washed once with Dulbecco's PBS (Cellgro, Mediatech, Inc., Herndon, VA). Whole cell extracts were made in radioimmunoprecipitation assay buffer (0.5% sodium deoxycholate, 1% NP40, 0.1% SDS, PBS, pH 7.4) Protein extracts, based on equivalent proteins, were fractionated by SDS-PAGE and transferred onto polyvinylidene fluoride membrane and immunoblotted with either an anti-Shh polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), anti-p21 (Santa Cruz Biotechnology), or anti-p53 monoclonal antibodies [a combination of Ab-2 (Neomarkers, Fremont, CA) and Ab-6 (Oncogene, San Diego, CA)], or anti-p63 (Santa Cruz Biotechnology), anti-myc (Zymed Laboratories, San Francisco, CA), anti-GST (Santa Cruz Biotechnology), or antiestrogen receptor (Lab Vision Corporation, Fremont, CA) antibodies for detection of GST-p63{gamma} or ER-p63{gamma}. Anti-p73 (Santa Cruz Biotechnology) and anti-ARF (Lab Vision Corporation) were used for the detection of p73 and p14ARF, respectively. Appropriate horseradish peroxidase–conjugated secondary antibodies (Promega) were used for chemiluminescence detection.

Transactivation Assays
Transactivation activity of the luciferase reporters was determined by performing luciferase assays (Promega) to measure Luciferase reporter activity and Bradford assays (Bio-Rad, Hercules, CA) to determine the protein levels. Transactivation activity was then calculated as relative luciferase units/protein concentration (RLU/protein) or RLU/ß-galactosidase activity for transfection efficiency normalization (pCMV-ß-galactosidase expression plasmid was used to control for transfection efficiency). For transactivation experiments using the ER-p63{gamma} fusion protein, 1 µmol/L of OHT was added to the cells to activate the protein.

RNA Isolation and TaqMan Reverse Transcriptase-PCR
For RNA studies, total RNA was extracted and TaqMan-based reverse transcription-PCR was done as described earlier (13) using assays on Demand reagents specific for human Shh, human p21, murine Shh, murine Id2 (PE Applied Biosystems, Foster City, CA).

ChIP Assay
H1299 cells grown in a 15 cm dish were transiently transfected with an expression plasmid encoding TAp63{gamma} for 20 hours. The ChIP assay was done using the ChIP kit and the manufacturer's recommended protocol (Active Motif, Carlsbad, CA) and done as previously described (13). PCR amplification of Shh promoter regions (sites 1-5) shown in Fig. 5E were done using 1.25 units of Pfx polymerase and a total of 100 pmol of primers per 50 µL reaction. Forty PCR cycles were done with each cycle consisting of 30 seconds at 94°C, 30 seconds at 58°C, and 1 minute at 68°C. The primers used for PCR amplification consisted of Shh site 1, sense 5'-GAGCTCTCTGTGCTTGATGACTGAAGC-3' and antisense 5'-CTCGAGTTGCTTTATTTTCACCTGAAG-3'; Shh site 2, sense 5'-GAGCTCAAG CCGGGAGTAACTGCTGT-3' and antisense 5'-CTCGAGAGAGAGGCTGCCTTTAGCACT-3'; Shh site 3, sense 5'-GAGCTCCAGCAGCAACAGAAAAAAAA-3' and antisense 5'-CTCGAGGGCTGGAATGGCAGGCTGCCG-3'; Shh site 4, sense 5'-GAGCTCCACAA GCTCTCCAGGCTTGC-3' and antisense 5'-CTCGAGTCCTCGCTCCGGCTCGCCCGC-3'; Shh site 5, sense 5'-GAGCTCCCTGTCTGGGTGGGGAGCGG-3' and antisense 5'-CTCGAGCTCGCCCATGGAACTGATGAC-3'. PCR products were resolved on a 2% agarose gel.


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank Drs. Steven Berberich and Patrick Dennis for critical review of the manuscript; and Shama Khokhar for technical assistance.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Grant support: American Cancer Society, Ohio Division Supported Research Grant.

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

Received 8/24/05; revised 7/22/06; accepted 8/15/06.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

  1. Yang A, Kaghad M, Wang Y, et al. p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities. Mol Cell 1998;2:305–16.[CrossRef][Medline]
  2. Yang A, Schweitzer R, Sun D, et al. p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 1999;398:714–8.[CrossRef][Medline]
  3. Mills AA, Zheng B, Wang XJ, Vogel H, Roop DR, Bradley A. p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature 1999;398:708–13.[CrossRef][Medline]
  4. Ishida S, Yamashita T, Nakaya U, Tokino T. Adenovirus-mediated transfer of p53-related genes induces apoptosis of human cancer cells. Jpn J Cancer Res 2000;91:174–80.[CrossRef]
  5. Donehower LA, Harvey M, Slagle BL, et al. Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature 1992;356:215–21.[CrossRef][Medline]
  6. Koster MI, Kim S, Mills AA, DeMayo FJ, Roop DR. p63 is the molecular switch for initiation of an epithelial stratification program. Genes Dev 2004;18:126–31.[Abstract/Free Full Text]
  7. Sasaki Y, Ishida S, Morimoto I, et al. The p53 family member genes are involved in the Notch signal pathway. J Biol Chem 2002;277:719–24.[Abstract/Free Full Text]
  8. Nguyen BC, Lefort K, Mandinova A, et al. Cross-regulation between Notch and p63 in keratinocyte commitment to differentiation. Genes Dev 2006;20:1028–42.[Abstract/Free Full Text]
  9. Carroll DK, Carroll JS, Leong CO, et al. p63 regulates an adhesion programme and cell survival in epithelial cells. Nat Cell Biol 2006;8:551–61.[CrossRef][Medline]
  10. Laurikkala J, Mikkola ML, James M, Tummers M, Mills AA, Thesleff I. p63 regulates multiple signalling pathways required for ectodermal organogenesis and differentiation. Development 2006;133:1553–63.[Abstract/Free Full Text]
  11. Ellisen LW, Ramsayer KD, Johannessen CM, et al. REDD1, a developmentally regulated transcriptional target of p63 and p53, links p63 to regulation of reactive oxygen species. Mol Cell 2002;10:995–1005.[CrossRef][Medline]
  12. Kim S, Han J, Kim J, Park C. Maspin expression is transactivated by p63 and is critical for the modulation of lung cancer progression. Cancer Res 2004;64:6900–5.[Abstract/Free Full Text]
  13. Kommagani R, Caserta TM, Kadakia MP. Identification of vitamin D receptor as a target of p63. Oncogene 2006;25:3745–51.[CrossRef][Medline]
  14. Osada M, Nagakawa Y, Park HL, et al. p63-specific activation of the BPAG-1e promoter. J Invest Dermatol 2005;125:52–60.[Medline]
  15. Sasaki Y, Naishiro Y, Oshima Y, Imai K, Nakamura Y, Tokino T. Identification of pigment epithelium-derived factor as a direct target of the p53 family member genes. Oncogene 2005;24:5131–6.[CrossRef][Medline]
  16. Yan W, Chen X. GPX2, a direct target of p63, inhibits oxidative stress-induced apoptosis in a p53-dependent manner. J Biol Chem 2006;281:7856–62.[Abstract/Free Full Text]
  17. Barbieri CE, Perez CA, Johnson KN, Ely KA, Billheimer D, Pietenpol JA. IGFBP-3 is a direct target of transcriptional regulation by {Delta}Np63{alpha} in squamous epithelium. Cancer Res 2005;65:2314–20.[Abstract/Free Full Text]
  18. Kaghad M, Bonnet H, Yang A, et al. Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers. Cell 1997;90:809–19.[CrossRef][Medline]
  19. Yang A, Walker N, Bronson R, et al. p73-deficient mice have neurological, pheromonal and inflammatory defects but lack spontaneous tumours. Nature 2000;404:99–103.[CrossRef][Medline]
  20. Pozniak CD, Barnabe-Heider F, Rymar VV, Lee AF, Sadikot AF, Miller FD. p73 is required for survival and maintenance of CNS neurons. J Neurosci 2002;22:9800–9.[Abstract/Free Full Text]
  21. Ho KS, Scott MP. Sonic hedgehog in the nervous system: functions, modifications and mechanisms. Curr Opin Neurobiol 2002;12:57–63.[CrossRef][Medline]
  22. Iseki S, Araga A, Ohuchi H, et al. Sonic hedgehog is expressed in epithelial cells during development of whisker, hair, and tooth. Biochem Biophys Res Commun 1996;218:688–93.[CrossRef][Medline]
  23. Freestone SH, Marker P, Grace OC, et al. Sonic hedgehog regulates prostatic growth and epithelial differentiation. Dev Biol 2003;264:352–62.[CrossRef][Medline]
  24. Liu S, Dontu G, Mantle ID, et al. Hedgehog signaling and Bmi-1 regulate self-renewal of normal and malignant human mammary stem cells. Cancer Res 2006;66:6063–71.[Abstract/Free Full Text]
  25. Shimada A, Kato S, Enjo K, et al. The transcriptional activities of p53 and its homologue p51/p63: similarities and differences. Cancer Res 1999;59:2781–6.[Abstract/Free Full Text]
  26. Osada M, Ohba M, Kawahara C, et al. Cloning and functional analysis of human p51, which structurally and functionally resembles p53. Nat Med 1998;4:839–43.[CrossRef][Medline]
  27. Bumcrot DA, Takada R, McMohan AP. Proteolytic processing yields two secreted forms of sonic hedgehog. Mol Cell Biol 1995;15:2294–303.[Abstract]
  28. Kitazawa S, Kitazawa R, Tamada H, Maeda S. Promoter structure of human sonic hedgehog gene. Biochim Biophys Acta 1998;1443:358–63.[Medline]
  29. Celli J, Duijf P, Hamel BC, et al. Heterozygous germline mutations in the p53 homolog p63 are the cause of EEC syndrome. Cell 1999;99:143–53.[CrossRef][Medline]
  30. Pomerantz J, Schreiber-Agus N, Liegeois NJ, et al. The Ink4a tumor suppressor gene product, p19Arf, interacts with MDM2 and neutralizes MDM2's inhibition of p53. Cell 1998;92:713–23.[CrossRef][Medline]
  31. Calabro V, Mansueto G, Santoro R, et al. Inhibition of p63 transcriptional activity by p14ARF: functional and physical link between human ARF tumor suppressor and a member of the p53 family. Mol Cell Biol 2004;24:8529–40.[Abstract/Free Full Text]
  32. Ronchini C, Capobianco AJ. Notch(ic)-ER chimeras display hormone-dependent transformation, nuclear accumulation, phosphorylation and CBF1 activation. Oncogene 2000;19:3914–24.[CrossRef][Medline]
  33. Kannan K, Amariglio N, Rechavi G, et al. DNA microarrays identification of primary and secondary target genes regulated by p53. Oncogene 2001;20:2225–34.[CrossRef][Medline]
  34. Osada M, Park HL, Nagakawa Y, et al. Differential recognition of response elements determines target gene specificity for p53 and p63. Mol Cell Biol 2005;25:6077–89.[Abstract/Free Full Text]
  35. Serber Z, Helen CL, Yang A, et al A C-terminal inhibitory domain controls the activity of p63 by an intramolecular mechanism. Mol Cell Biol 2002;22:8601–11.[Abstract/Free Full Text]
  36. Senoo M, Matsumura Y, Habu S. TAp63{gamma} (p51A) and dNp63{alpha} (p73L), two major isoforms of the p63 gene, exert opposite effects on the vascular endothelial growth factor (VEGF) gene expression. Oncogene 2002;21:2455–65.[CrossRef][Medline]
  37. Wu G, Osada M, Guo Z, et al. {Delta}Np63{alpha} up-regulates the Hsp70 gene in human cancer. Cancer Res 2005;65:758–66.[Abstract/Free Full Text]
  38. Wu G, Nomoto S, Hoque MO, et al. {Delta}Np63{alpha} and TAp63{alpha} regulate transcription of genes with distinct biological functions in cancer and development. Cancer Res 2003;63:2351–7.[Abstract/Free Full Text]
  39. Jacobs WB, Govoni G, Ho D, et al. p63 is an essential proapoptotic protein during neural development. Neuron 2005;48:743–56.[CrossRef][Medline]
  40. Kadakia M, Slader C, Berberich SJ. Regulation of p63 function by Mdm2 and MdmX. DNA Cell Biol 2001;20:321–30.[CrossRef][Medline]
  41. Zeng X, Goetz JA, Suber LM, Scott WJ, Jr., Schreiner CM, Robbins DJ. A freely diffusible form of Sonic hedgehog mediates long-range signalling. Nature 2001;411:716–20.[CrossRef][Medline]



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