Carcinogenesis

Carcinogenesis Advance Access originally published online on August 4, 2005
Carcinogenesis 2006 27(1):53-63; doi:10.1093/carcin/bgi200

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Carcinogenesis vol.27 no.1 Published by Oxford University Press 2005.

Unique domain functions of p63 isotypes that differentially regulate distinct aspects of epidermal homeostasis

K.E. King, R.M. Ponnamperuma, M.J. Gerdes ¹, T. Tokino ², T. Yamashita ², C.C. Baker ¹ and W.C. Weinberg ^*

Center for Drug Evaluation and Research, FDA, Bethesda, MD 20892, USA, ¹ National Cancer Institute, NIH, Bethesda, MD 20892, USA and ² Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan

^* To whom correspondence should be addressed at: Division of Monoclonal Antibodies, Office of Biotechnology Products, Center for Drug Evaluation and Research, FDA, 29B Lincoln Drive, HFD-123, Building 29B, Room 3NN04, Bethesda, MD 20892-4555, USA. Tel: +1 301 827 0709; Fax: +1 301 827 0852; Email: weinberg{at}cber.fda.gov

	Abstract

Top Abstract Introduction Materials and methods Results Discussion Supplementary material References

 
p63 is critical for squamous development and exists as multiple isotypes of two subclasses, TA and N. Np63 isotypes can antagonize transcription by TAp63 and p53, and are highly expressed in squamous cell cancers. Using mouse keratinocytes as a biological model of squamous epithelium, we show that multiple p63 isotypes, N- and TA-containing, are expressed and differentially modulated during in vitro murine keratinocyte differentiation. Np63 declines with Ca²⁺-induced differentiation, while a smaller N-form, Np63_s, persists, suggesting unique functions of the two N-forms. To investigate the impact of dysregulated p63 expression that is observed in cancers and to define the biological contribution of the different domains of the p63 isotypes, Np63, Np63^p40, TAp63, TAp63 or ß-galactosidase were overexpressed in primary murine keratinocytes. Microarray, RT–PCR and western blot analyses revealed that overexpression of Np63^p40, which lacks the entire -tail present in Np63, permits expression of a full panel of differentiation markers. This is in contrast to overexpression of the full-length Np63, which blocks induction of keratin 10, loricrin and filaggrin. These findings support a role for the -tail of Np63 in blocking differentiation-specific gene expression. Overexpression of either TAp63 isotype permits keratin 10 and loricrin expression, thus the -terminus requires the cooperation of the N domain in blocking early differentiation. However, both TA isotypes block filaggrin induction. The N-terminus is sufficient to maintain keratinocytes in a proliferative state, as both N forms block Ca²⁺-mediated p21^WAF1 induction and S-phase arrest, while sustaining elevated PCNA levels. No alteration in cell cycle regulation was observed in keratinocytes overexpressing TAp63 or TAp63. Clarifying the functional distinctions between p63 isotypes and domains will help to elucidate how their dysregulation impacts tumor biology and may suggest novel therapeutic strategies for modulating behavior of tumor cells with altered expression of p53 family members.

Abbreviations: Ad-Np63, overexpression of Np63 mediated by adenovirus; ß-gal, ß-galactosidase; BrdU, bromodeoxyuridine; FACS, fluorescence activated cell sorting; MOI, multiplicity of infection

	Introduction

Top Abstract Introduction Materials and methods Results Discussion Supplementary material References

 
The p53 homologue p63 exists as multiple protein isotypes of two subclasses, TA and N, which arise as a result of alternative promoter usage (1) (Figure 1A). TAp63 isotypes contain a transactivation domain homologous to that of p53 and display overlapping transcriptional activity with p53. In contrast, Np63 isotypes do not contain this domain and are capable of antagonizing transcriptional activity mediated by p53 as well as by TAp63 isotypes (1). Although mutation of p63 is not common in human cancers, the p63 gene locus is amplified in squamous cell carcinomas of the head and neck (2–5). Overexpression of p63 protein and mRNA, particularly Np63, has been reported in a variety of squamous cell cancers including those of the head and neck, lung, and skin (5–12). Expression of Np63 isotypes has been associated with undifferentiated cells of proliferative potential in both normal epidermis and tumors (13). We have shown that in vitro overexpression of Np63 in primary murine keratinocytes blocks Ca²⁺-induced growth arrest and differentiation (14). Combined with the expression patterns reported in cancers, this observation suggests a role for Np63 in the maintenance of the basal cell phenotype in normal and neoplastic epithelium.

View larger version (39K): [in this window] [in a new window]   Fig. 1. N and TAp63 isotypes are expressed in primary murine keratinocytes. (A) Graphic depicting the TA and Np63 isotypes. Exon usage is noted next to the isotypes. Exon 3' is the first exon of Np63 mRNA. Domains noted are: TA, transactivation; DBD, DNA binding; OD, oligomerization; SAM, sterile -motif. (B) Semi-quantitative RT–PCR analysis of mRNA isolated from proliferating (0.05 mM Ca2+) or differentiating (0.12 mM Ca2+) primary murine keratinocyte cultures 24 h after inducing keratinocyte differentiation via increasing extracellular [Ca2+]. Primers are located within corresponding 5' and 3' domains. HPRT is a control for quantitation. Reactions without reverse transcriptase (–RT) were included as negative controls. mRNA expression levels of Np63 and Np63 decrease with differentiation relative to proliferating cultures, whereas TAp63 levels increase. No evidence of either TAp63 or TAp63ß expression was detected (results not shown). See online Supplementary material for a color version of this figure.

 
p63 expression has been reported to progressively increase from the pre-neoplastic lesion to invasive squamous cell carcinoma of the lung (12,15). Reports of TAp63 isotypes in tumors are few, however a trend for decreased TAp63 levels has been correlated with a poor clinical outcome in buccal and laryngeal squamous cell carcinomas (11,16). Co-expression of the N and TAp63 isotypes occurs in normal tissues (17), and in a given cellular context the relative ratios of these isotypes could ultimately affect biological outcome.

Additional variations within the p63 subclasses are the result of C-terminal alternative splicing. , ß and variants, which incorporate varying portions of the gene sequence at their C-termini, have been described for both TA and Np63 isotypes (Figure 1A) (18,19). The -variants are the longest and contain a SAM domain, which is a putative protein–protein interaction domain (20). A second transactivation domain and a transactivation inhibitory domain have been described C-terminal to the SAM domain (21,22). Within the context of the TAp63 subclass, the -terminus has been reported to auto-inhibit transcriptional activity (22,23). ß-Splice variants lack the SAM domain but retain the second transactivation domain, and lose auto-inhibitory activity (22). -variants lack the C-terminal exon sequences specific to the and ß variants, but incorporate additional sequences of unknown function from exon 15. The shortest isotype described, Np63^p40, is a further N variant that truncates immediately after the oligomerization domain (4).

In this study we used primary murine keratinocytes as a model of squamous epithelium to address the following questions: How is p63 isotype expression altered during normal keratinocyte differentiation? What are the biological roles of the endogenously expressed forms? What are the specific contributions of the TA and N domains, and how does the C-terminus influence p63 function? To address the latter points, we evaluated the consequences of overexpressing either of two endogenously expressed p63 isotypes (Np63, TAp63), or two C-terminal variants of these isotypes (Np63^p40, TAp63), on keratinocyte proliferation and differentiation. Our findings provide insight into the biological contribution of different functional domains of p63 isotypes and how dysregulation of p63, as observed in squamous cell carcinomas, might contribute to cancer pathogenesis.

	Materials and methods

Top Abstract Introduction Materials and methods Results Discussion Supplementary material References

 
Cell culture
Primary keratinocytes were isolated from the skin of 1–2-day-old C57Bl/6NCr mice and cultured as described previously (24,25). Standard growth medium was composed of Minimal Essential Medium (SMEM) (Life Technologies, Gaithersburg, MD), 8% chelexed fetal bovine serum (FBS) (Gemini BioProducts, Calabasas, CA), 10 U/ml penicillin and 10 ug/ml streptomycin (Life Technologies), at a final concentration of 0.05 mM Ca²⁺. Differentiation was induced by elevating extracellular [Ca²⁺] to 0.12 mM (24). Similar patterns of endogenous p63 expression were observed in primary keratinocytes derived from either C57Bl/6NCr or Sencar mice.

Immunostaining
Ethanol-fixed samples of chemically induced mouse skin tumors derived from two-stage carcinogenesis protocols were embedded in paraffin and incubated with peroxidase blocking buffer (KPL, Gaithersburg, MD) followed by pan-p63 antibody (4A4, Santa Cruz Biotechnology, Santa Cruz, CA), or antibodies specific for the N-terminus (p40 Ab-1, Oncogene Research Products, Boston, MA) or C-terminus (H-129, Santa Cruz Biotechnology). Sections were then incubated with biotinylated secondary antibodies to rabbit or mouse IgG, as appropriate, followed by streptavidin–horseradish peroxidase conjugate, and binding was visualized with Histomark Orange Detection System as per manufacturer's directions (KPL). Nuclei were counterstained with contrast green (KPL).

Adenoviruses and adenoviral infections
Adenovirus encoding Np63 was generated as described previously (14). Adenoviruses encoding TAp63, TAp63, p53 and ß-galactosidase (ß-gal) were previously described (26) and adenovirus encoding Np63^p40 (5) was the generous gift of Dr D.Sidransky.

For adenoviral mediated gene transduction, keratinocytes were incubated 60 min at 37°C in serum-free growth medium with an equal MOI of one of the following adenoviral constructs: ß-gal, Np63, TAp63, TAp63, Np63^p40 or p53. Following the incubation, the medium was aspirated and replaced with SMEM, described above. Expression was verified by western analysis with an antibody that recognizes the p63 core DNA-binding domain.

Western analysis
Soluble and total cell lysates for western analysis were prepared as described previously (14,27). Optitran nitrocellulose membranes (Schleicher and Schuell, Keene, NH) were probed with antibodies recognizing the following of both mouse and human p63: p63 core DNA binding domain (4A4, Santa Cruz Biotechnology or OP132, Oncogene Research Products); p63 TA, and domains (D20, C-18, H129 and N-18, respectively, Santa Cruz Biotechnology); p63 N domain (p40 Ab-1, Oncogene Research Products); actin (AC-15, Sigma Immuno Chemicals, St Louis, MO); p21^WAF1 and PCNA (OP79 and NA03, Oncogene Research Products); keratins 1, 10, 14 and filaggrin (BABCO, Richmond, CA). Signal was detected using HRP-linked anti-mouse (1:5000), anti-goat (1:10000) or anti-rabbit (1:1000) secondary antibody as appropriate and enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech, Piscataway, NJ). Results shown are representative of a minimum of two independent experiments.

Fluorescence activated cell sorting (FACS) analysis
Confluent cultures of primary murine keratinocytes were adenovirally infected with Ad-TAp63, Ad-TAp63, Ad-Np63^p40 or Ad-ß-gal at 1.5 days post-plating and FACS analysis was performed as described previously (14). Briefly, 17 h post-infection the medium was changed and cells were maintained for 24 h in 0.05 mM or 0.12 mM Ca²⁺. Cultures were pulsed with 10 µM bromodeoxyuridine (BrdU) for the final 4 h. Cell suspensions were fixed in 70% ethanol for 24 h and incubated with an antibody to BrdU (Becton-Dickinson, San Jose, CA) as per the manufacturer's protocol, then washed and resuspended in phosphate buffered saline containing propidium iodide (5 µg/ml). The cell cycle distribution was analyzed using Cell Quest software on a Becton Dickinson FACSCalibur. A minimum of three replicates were analyzed per condition. Results from each of three independent experiments are presented as the mean percentage of cells in S-phase per condition ± standard deviation.

RNA isolation and semi-quantitative RT–PCR for markers of keratinocyte differentiation
Total RNA was isolated from primary murine keratinocytes using an RNAqueous Kit (Ambion, Austin, TX). Contaminating DNA was removed from 10 µg of total RNA by incubation with 5 U DNase I for 1 h at 37°C in a total volume of 100 µl. The reaction was stopped by incubation with 1 µl of stop solution at 65°C for 10 min and the RNA precipitated with 0.1 vol 3 M NaAc (pH 5.2) and 2.2 vol 100% ethanol. RNA (5 µg) was reverse transcribed according to the instructions provided in the SuperScript First Strand Synthesis System for PCR Kit (Invitrogen Life Technologies, Carlsbad, CA) and included 50 ng each of random hexamers and oligo(dT)_12–18 primer.

Aliquots (2.5%) of the cDNA pools generated by reverse transcription were used as templates in amplification reactions to detect differentiation-specific gene expression. Target sequences were amplified in a reaction mixture containing 1x reaction buffer (10 mM Tris–HCl (pH 8.3), 50 mM KCl and 1.5 mM MgCl₂), 400 µM each dNTP (10 mM mix), 5 U AmpliTaq DNA polymerase (all from Applied Biosystems) and 250 ng each primer. Following a 5 min hot start at 94°C, the reaction profile was as follows: denaturation at 94°C for 45 s; annealing (K10: 58°C, 33 cycles; K1: 58°C, 27 cycles; loricrin: 56°C, 30 cycles; filaggrin: 61°C, 30 cycles and GAPDH: 60°C, 25 cycles) for 45 s; elongation at 72°C for 1 min. The following primer sequences were used, and expected product sizes are noted in parentheses:

Keratin 10: 5'-gaatcgcaaggatgctgaag-3' and 5'-tctccagtcgggtcttgatg-3' (338 bp);

Keratin 1: 5'-gcaagaccaagatcaatcccac-3' and 5'-aaattaaggcggctcagcg-3' (356 bp);

Loricrin: 5'-tacctggccgtgcaagtaag-3' and 5'-aacaggatacacctggagcgac-3' (181 bp);

Filaggrin: 5'-gccaagtccattctggagtc-3' and 5'-ctactgcctggccttctgag-3' (306 bp);

GAPDH: 5'-tgttcctacccccaatgtgtc-3' and 5'- tctcttgctcagtgtccttgc-3' (352 bp).

To achieve greater accuracy, PCR profiles were initially determined over a range of cycles for each primer pair, so that reactions could be stopped before saturation. All RT–PCRs were performed in duplicate or triplicate and results shown are representative of at least two independent RNA preparations.

Semi-quantitative RT–PCR for p63 isotype expression
RNA was isolated and DNase I treated as described above. Total RNA (0.5 µg) was reverse transcribed with Accuscript Reverse Transcriptase (Stratagene, La Jolla, CA) according to the manufacturer's instructions. The reaction mixture included 0.5 µg oligo(dT)₁₈ primer.

Aliquots (5%) of the cDNA pools generated by reverse transcription were used as templates in amplification reactions to detect p63 isotype expression. Target sequences were amplified in a reaction mixture that included heat activated Herculase Hotstart DNA polymerase, as per manufacturer's instructions (Stratagene). Following a 3 min hot start at 94°C, reaction profiles were as follows: denaturation at 94°C for 30 s; annealing at 53°C (TAp63, Np63 and TAp63/ß), 54°C (Np63), or 51°C (HPRT) for 30 s; extension 72°C for 1.5 min. The number of cycles performed for each primer set was: 35 (TAp63, Np63, Np63 and TAp63/ß) or 25 (HPRT). The primer recognizes the exon 12–13 splice junction and is therefore specific for the –terminus. The /ß primer recognizes C-terminal sequences present in exon 12, which are present in both and ß p63 isotypes. Primer sequences were as follows (F: forward; R: reverse):

TA: 5'-ATG TCG CAG AGC ACC CAG-3' (F)

(28): 5'-CTC CAC AAG CTC ATT CCT GAA GC-3' (R)

N (28): 5'-CCA GAC TCA ATT TAG TGA GCC AC-3' (F)

: 5'-ACA ACC TTG CTA AGA AAA CTG A-3' (R)

/ß (28): 5'-ATT GCG CTG CTG TGG GTT GAT AAG-3' (R)

HPRT (28): 5'-CGT CGT GAT TAG CGA TGA TGA-3' (F)

HPRT (28): 5'-TTC AAA TCC AAC AAA GTC TGG C-3' (R)

All RT–PCRs were performed in duplicate or triplicate and results shown are representative of at least two independent experiments. All experiments included reactions in which RT was omitted as negative controls, and positive size controls derived from amplification reactions using murine p63 isotype cDNA constructs as templates. The identity of PCR products was confirmed by restriction enzyme mapping or nested PCR.

Real-time PCR for quantitation of specific p63 domains
Real-time PCR was performed on RNA samples prepared, DNase I treated and reverse transcribed with Superscript First Strand Synthesis System as for keratinocyte markers, with the exception that 100 ng random hexamers/µg of RNA were utilized. Isoform specific PCR assays were designed so that one primer spanned an exon–exon boundary (29). The exons bridged in each reaction are noted below and the primer that bridges the exon–exon junction is identified by a ^*.

N (exons 3' and 4): ^*5'-cagactcaatttagtgagcc-3' and 5'-ctgctggtccatgctgtt-3'

(exons 13 and 14): 5'-cagacttgccaaatcatcc-3' and ^*5'-cagcattgtcagtttcttagc-3'

(exons 10 and 15): 5'-agatcaaagagtcactggagc-3' and ^*5'-caggctgaaaggagatgt t-3'

This approach allows quantification of the N subclass and the specific and splice variants. However, it does not allow direct determination of which subclass, N or TA, the or splice variants belonged.

Levels of 18S RNA were quantified using primers 5'-gccctgtaattggaatgag-3' and 5'-tatacgctattggagctgga-3' for the purpose of normalizing efficiency of RT reactions between RNA preparations. Keratin 1 levels were also monitored to provide a positive biological control and confirm suitability of the culture conditions (24), using the following primers: 5'-agctacggtggctcctct-3' and 5'-ctggtggaaacaaacttcac-3'.

PCR was carried out using the Perkin Elmer SYBR green real-time PCR kit. Reactions and data collection were performed on a Bio-Rad iCycler iQ Real-time PCR Detection System (Bio-Rad, Hercules, CA). Negative controls (TE, mouse genomic DNA, and negative murine cDNA isotype) were included in every assay. A standard curve using the appropriate cloned murine cDNA standard was set up to accompany each reaction to allow for absolute quantification of each isotype. N and reactions both utilized the same Np63 cDNA standard and a TAp63 standard was used for the quantification of the domain. An additional real-time PCR assay for the DNA binding domain, which is common to these two isotypes, was used to ensure the accuracy of the quantitation of these two standards relative to each other. This allows data for all isotypes to be expressed in terms of fg of the cDNA clone for Np63. Thus, all data may be directly and quantitatively compared. Two-step PCR was carried out with data collection and analysis during the combined annealing and extension step. Denaturation was at 95°C for 15 s and anneal/extension at 60°C for 1 min. Three separate mRNA preparations were evaluated and each mRNA sample was assayed in triplicate. All data were converted to fg of the Np63 cDNA standard. The 18S data were used to generate a normalization factor, which was set to 1 for the 0.05 mM Ca²⁺ sample. This normalization factor was applied to all isotype data to correct for any differences in the efficiency of the reverse transcriptase step and errors in RNA quantitation.

Microarray
To assess the contribution of the -tail to Np63 function, cDNA microarray analysis was performed on RNA samples isolated with TRIzol reagent (Life Technologies) from keratinocytes overexpressing Ad-Np63, Ad-Np63^p40 or Ad-ß-gal (as a control) cultured under 0.12 mM Ca²⁺ conditions. Np63 versus Np63^p40; Np63 versus ß-gal; Np63^p40 versus ß-gal sample pairs were analyzed using Cy3 and Cy5 dyes on mouse cDNA arrays custom synthesized at the National Cancer Institute, NIH. To confirm accuracy and to avoid bias owing to Cy3 or Cy5 dye, labeling reactions were performed in reverse for each paired set. Each final dataset contained four independent hybridizations from two separate RNA preparations. Labeling, hybridization and detection were carried out with the Genisphere sub-Micro labeling kit according to manufacturer's instructions (Genisphere, Hatfield, PA). Slides were scanned on an Axon 4000B scanner and PMT voltage levels were adjusted to obtain an equivalent overall intensity for Cy3 and Cy5. Scanned images were then processed with Genepix 4.0 software to quantitate spot intensities for each label. Spots with high artifactual background or no signal were flagged for exclusion. The intensity files were uploaded and analyzed with the mAdb software suite (CIT, NCI, NIH, Bethesda, MD). The following parameters were set for analysis: Target spot size 100–400 µm and 2-fold difference in three of four arrays (75%). Values for the reverse labeling were reciprocated. A table was then generated including the specific spot images with the list of outliers and the respective intensity ratio differences. The spot images were further examined and inadequate spots were excluded. Output was sorted based on ratio values and is presented as mean fold increase.

	Results

Top Abstract Introduction Materials and methods Results Discussion Supplementary material References

 
N and TAp63 isotypes are differentially expressed during keratinocyte differentiation
p63 isotypes can antagonize each other functionally (1) and this modulation may serve to mediate the effects of individual p63 isotypes within both normal and neoplastic environments. Using primary murine keratinocytes as a model of squamous epithelium, we characterized p63 isotype expression patterns at both the mRNA and protein levels in proliferating keratinocyte cultures and after induction of terminal differentiation via the modulation of the extracellular [Ca²⁺] in the medium from 0.05 to 0.12 mM Ca²⁺ for 24 h.

Semi-quantitative RT–PCR analysis demonstrated readily detectable levels of mRNA for Np63 and Np63 in proliferating cultures, and very low levels of TAp63 (Figure 1B). mRNA levels of both Np63 isoforms decline in cultures of differentiating keratinocytes, whereas TAp63 mRNA levels increase. We found no evidence for expression of mRNA for TAp63 or TAp63ß (not shown). The p63 mRNAs observed in these cultures are consistent with those recently reported in intact mouse skin (28). To correlate mRNA and protein levels, we performed western analysis using a pan-p63 antibody that recognizes the core DNA binding domain of p63 as well as domain-specific antibodies directed to either N, TA, or . This revealed a complex pattern of p63 isotype modulation during keratinocyte maturation in vitro (Figure 2A). Differential expression and modulation of multiple p63 isotypes was observed following reactivity with a pan-p63 antibody (Figure 2A). The identity of two of these isotypes, Np63 and TAp63, was established with the use of domain-specific antibodies (Figure 2B) and confirmed with size standards (Figure 2C). Np63 is the predominant isotype expressed in proliferating keratinocytes and is recognized by the pan-p63 antibody as well as N and antibodies. The expression pattern observed with all three of these antibodies demonstrates that levels of Np63 decline in maturing keratinocytes (Figure 2A and B, N and -panels). These results are consistent with the RT–PCR analysis shown in Figure 1 and previous reports (1,13,14,30). In contrast, in multiple independent experiments the level of TAp63 protein observed in proliferating cultures is sustained or upregulated as keratinocytes differentiate, but never decreases (Figure 2A, pan-p63; Figure 2B, TA and -panels). The increase in TAp63 protein with differentiation correlates with mRNA levels for TAp63 observed by semi-quantitative RT–PCR (Figure 1B). Controls to confirm the specificity of the domain-specific antibodies are shown in Figure 2D.

View larger version (41K): [in this window] [in a new window]   Fig. 2. Differential expression of N and TAp63 isotypes during keratinocyte differentiation. Protein levels of specific p63 isotypes are independently regulated during differentiation. (A and B) Endogenous levels of p63 were evaluated by western analysis of samples prepared from proliferating (0.05 mM Ca2+) or differentiating (0.12 mM Ca2+) keratinocytes 24 h after inducing keratinocyte differentiation via increasing extracellular [Ca2+]. (A) The pan-p63 antibody recognizes sequences within the core p63 DNA binding domain and thus reacts with multiple p63 isotypes. Identities of Np63 and TAp63 were confirmed by the use of domain-specific antibodies (B) and by co-migration with size standard controls (C). Np63 expression declines during differentiation, while TAp63 remains stable or is upregulated. An additional N variant (Np63s) is recognized by the pan-p63 and N antibodies and is stably expressed or increases. The increase seen in Np63s levels in panel A may reflect the presence of an additional band in 0.12 mM cultures that is recognized by the pan-p63 but not the N antibody. The lysates used in A and B were derived from independent primary keratinocyte preparations. (C and D) cDNA controls as size standards for Np63p40, Np63, TAp63, Np63 and TAp63 were prepared by transfection or adenoviral transduction of primary murine keratinocytes and probed with a panel of p63 antibodies. Controls are shown next to primary murine keratinocyte lysate prepared from proliferating (C) or differentiating (D) keratinocytes. TAp63 and Np63 co-migrate with the appropriate size controls. Np63s migrates between Np63p40 and Np63. (D) A single isotype corresponding to TAp63 is recognized in the murine keratinocyte lysate by the TA specific antibody, whereas two isotypes are recognized in the primary murine keratinocyte lysate by the N antibody. These isotypes correspond to Np63s and Np63 identified on the pan-p63 blot. The middle band observed in the N blot does not co-migrate with any bands recognized by the pan-p63 antibody, and is, therefore, believed to be non-specific. The -specific antibody detected a single -isotype (endogenous and control). The -specific antibody detects both Np63 and TAp63 size controls, but only TAp63 is detected in the primary murine keratinocyte lysate. (E) Real-time RT–PCR analysis performed on mRNA samples prepared from proliferating (0.05 mM Ca2+) or differentiating (0.12 mM Ca2+) keratinocytes 24 h after inducing keratinocyte differentiation via increasing extracellular [Ca2+]. mRNA expression of Np63 and the C-terminal and splice variants decreases with differentiation (left panel). Higher levels of N-domain expression than the combined - and -domains support the existence of a N isotype in addition to Np63 and Np63. Results are presented as mean fg amounts of mRNA specific for each domain ± SD. Analysis was performed on three independent RNA preparations in triplicate; graph of representative experiment presented. Keratin 1 mRNA levels were monitored to provide a positive control and confirm suitability of culture conditions (right panel).

 
A third strong band detected with the pan-p63 antibody and noted in Figure 2A was identified as a member of the N subclass based on an N-terminal specific antibody (Np63_s, Figure 2A and B). Co-migration next to a Np63^p40 size standard revealed that this shorter N isotype (Np63_s) migrates between human Np63^p40 and murine Np63 (Figure 2C and D, N antibody). It is not recognized by the antibody (Figure 2B), suggesting that it is not Np63. Furthermore, the pan-p63 and N western blots did not reveal a species co-migrating with the Np63 size control (Figure 2C and D). Based on the contrasting results between the RT–PCR and western analyses, it appears that the high levels of Np63 transcript seen by RT–PCR are not translated in primary murine keratinocytes or that the translated protein is very unstable (Figures 1 and 2), and that the additional N band is unique from Np63.

Unlike Np63, Np63_s persists or increases following induction of terminal differentiation (Figure 2A and B, N, pan-p63 panels). Its relative size as well as the failure of this band to react with a -specific antibody (Figure 2B, panel) suggests that it is a novel N form, however we cannot exclude the possibility that it is the previously undescribed murine p40 homologue that could have undergone post-translational modification.

The presence of a N isotype in addition to Np63 is supported by quantitative RT–PCR data (Figure 2E). Real-time PCR primers were designed to measure the absolute amounts of N subclass and or specific splice variants. The amount of mRNA for Np63 is substantially more than that measured for and domains combined, consistent with expression of an additional N isotype. Overall, mRNA for Np63 declines with keratinocyte differentiation, as do expression levels of both C-terminal splice variants tested, and (Figure 2E). Induction of keratin 1 mRNA confirmed the suitability of the culture conditions (Figure 2E, right panel).

These results demonstrate that several p63 isotypes are co-expressed in keratinocytes, and their balance is altered as keratinocytes differentiate.

Np63 is highly expressed in chemically induced tumors of mouse skin
As shown in Figure 3A, immunohistochemical analysis of chemically induced papillomas of mouse skin using the 4A4 pan-p63 antibody revealed a basilar expression pattern, consistent with previous reports of p63 staining in human squamous cell skin tumors (13,31). Similarly, reactivity of well-differentiated carcinomas with this antibody is localized to keratinocytes removed from areas of differentiation (Figure 3B). and N domain specific antibodies yielded similar expression patterns to 4A4, consistent with Np63 expression (Figure 3C and D). A more uniform staining pattern throughout the tumor was seen in undifferentiated carcinomas and was similar to all three antibodies (not shown). Only contrast green counterstain was detected in parallel control sections treated identically but without primary antibody (not shown).

View larger version (61K): [in this window] [in a new window]   Fig. 3. Np63 is highly expressed in squamous cell tumors. Immunostaining of sections derived from chemically induced tumors of mouse skin. (A and B) Tumor sections incubated with 4A4 pan-p63 antibody show nuclear localization of p63 within basal cells of papillomas (A) and in undifferentiated portions of carcinomas (B). (C and D) Neighboring sections of the well-differentiated carcinoma, as shown in B, reacted with N and specific antibodies. In all cases, p63 staining is localized to keratinocytes removed from centers of differentiation. Results are consistent with high levels of Np63 in these tumors. See online Supplementary material for a color version of this figure.

 
Np63 isotypes differ in their ability to block keratinocyte differentiation
Previous studies have shown that overexpression of Np63 in keratinocytes prevents the induction of differentiation markers keratin 10 and filaggrin (14). Although the N domain is believed to confer dominant negative properties on this class of isotypes, distinct functions are inferred by the differential expression of full length Np63 and the abbreviated Np63_s isotype. To elucidate these functional distinctions as well as a potential role of the -tail, microarray analysis was performed on keratinocytes under differentiating conditions overexpressing either full-length Np63 or Np63^p40. At least two sets of direct comparisons were made between the following samples: Np63 versus Np63^p40, Np63 versus ß-gal and Np63^p40 versus ß-gal. Strikingly, a number of genes whose expression has been associated with keratinocyte differentiation were noted to be upregulated at least 2-fold in keratinocytes overexpressing Np63^p40 relative to Np63 (Table I) (24,32–36), whereas a similar distinction was not observed between keratinocytes overexpressing Np63^p40 versus ß-gal (data not shown). In contrast, an enhancement of markers associated with basal cells was observed in keratinocytes overexpressing Np63 relative to both Np63^p40 and ß-gal (Table I) (37–40), consistent with our previous finding that Np63 overexpression blocks keratinocyte differentiation (14). These findings suggested that, in contrast to Np63 (14), overexpression of Np63^p40 permits keratinocyte differentiation. This was confirmed by RT–PCR and western analysis of markers of differentiation-specific gene expression (Figures 4A and 5). Np63^p40-overexpressing keratinocytes demonstrate normal Ca²⁺-mediated induction of the full range of differentiation-specific proteins evaluated, including keratins 1 and 10 and filaggrin (Figure 4A). Likewise, normal induction of mRNA for these markers, as well as loricrin mRNA, was observed in Np63^p40-overexpressing cells using RT–PCR (Figure 5). The previously described block in induction of keratin 10 and filaggrin by Np63 (14) is mediated at the RNA level and is also observed for loricrin (Figure 5, top panels). These results suggest that the -tail of Np63 plays an important role in abrogating expression of differentiation-specific genes.

View this table: [in this window] [in a new window]   Table I. Microarray analyses of keratinocytes overexpressing Np63p40 or Np63 indicate altered differentiation status of the two populations

 

View larger version (70K): [in this window] [in a new window]   Fig. 4. p63 isotypes exert distinct effects on the keratinocyte differentiation program. Keratinocytes were infected with adenovirus encoding human Np63p40, TAp63, TAp63, p53 or ß-galactosidase (ß-gal), as noted at the top of each panel. Seventeen hours post-infection cultures were either maintained in medium containing 0.05 mM Ca2+ or induced to differentiate by increasing extracellular [Ca2+] to 0.12 mM. Whole cell lysates were harvested 8, 24 or 32 h post-Ca2+ trigger, as noted. (A, B, C and D) Top panels: Membranes were probed sequentially with antibodies directed to filaggrin, keratin 10 and keratin 14. Bottom panels: Replicate western blots of lysates described above were probed sequentially with antibodies directed to keratin 1, ß-gal, p63 (pan-p63) or p53, and keratin 14. For each antibody within an experiment, the ß-gal control and test sample blots were exposed to ECL reagent and film for the same length of time. Panels probed with ß-gal and p53/p63 antibodies are included to demonstrate adenoviral driven expression of these genes. The p63 bands shown in these panels correspond to the specific p63 splice variant introduced by adenoviral transduction. Overexpression of Np63p40 or p53 allowed for full expression of differentiation markers. Both TAp63 and TAp63 interfere specifically with the induction of filaggrin, a late marker of differentiation.

 

View larger version (73K): [in this window] [in a new window]   Fig. 5. Effects of p63 isotypes on differentiation-specific gene expression are regulated at the RNA level. Keratinocytes were infected with adenovirus encoding Np63, Np63p40, TAp63, TAp63, p53 or ß-gal. Seventeen hours post-infection cultures were either maintained in medium containing 0.05mM Ca2+ or induced to differentiate by increasing extracellular [Ca2+] to 0.12 mM. RNA samples were harvested 24 h later, and semi-quantitative RT–PCR was performed using primers specific for keratin 1, keratin 10, loricrin, filaggrin and GAPDH. Induction of differentiation markers at the RNA level paralleled the results seen by western blotting. Overexpressed Np63 blocked keratin 10 and filaggrin, but not keratin 1, consistent with the protein expression patterns previously described (14). Both TAp63 and TAp63 blocked expression of filaggrin mRNA.

 
TAp63 isotypes block filaggrin expression but not early markers of differentiation
To determine whether the -tail impacts differentiation outside the context of the N subclass of isotypes, TAp63 was introduced via adenoviral transduction and effects on markers of differentiation were evaluated by western analysis and RT–PCR. In contrast to our previous results with Np63, TAp63 permitted keratin 10 induction at both the protein (Figure 4B) and mRNA (Figure 5) levels. Thus, the presence of the -terminus is not sufficient to block early differentiation, but acts only in conjunction with the N-terminus.

As endogenous expression of TAp63 is modulated with differentiation, we investigated whether an imbalance in the TAp63 isotype would influence the keratinocyte differentiation response to elevated [Ca²⁺]. Similar to TAp63, TAp63 had no effect on the induction of keratins 1 and 10 (Figure 4B and C). However, both TA isotypes blocked induction of filaggrin protein (Figure 4B and C). RT–PCR analysis for differentiation-specific markers revealed that this block in filaggrin by both TAp63 isotypes is mediated at the RNA level (Figure 5). These findings suggest that the TA domain has a specific effect on filaggrin expression. Overexpression of p53, which shares 22% homology with the p63 TA domain, has no discernible effect on differentiation marker expression (Figures 4D and 5).

Both Np63 isotypes block Ca²⁺-induced growth arrest
The ability of Np63 to abrogate growth arrest mediated by elevated Ca²⁺ has been demonstrated previously (14). Despite differences between the Np63 isotypes with regard to differentiation potential, both Np63 and Np63^p40, when overexpressed, impair the Ca²⁺-induced growth arrest that is associated with differentiation. As shown in Figure 6A, keratinocytes overexpressing Ad-ß-gal demonstrate a 37% decrease in the S-phase population under 0.12 mM Ca²⁺ conditions, whereas cells overexpressing Np63^p40 show only an 11% decrease in S-phase population. Overexpressing Ad-TAp63 or Ad-TAp63 does not alter the Ca²⁺-induced growth arrest, with S-phase population reductions in 0.12 mM Ca²⁺ conditions of 37 and 45%, similar to that observed in the control cultures (Figure 6A). The decrease in S-phase cells in ß-gal control cultures following Ca²⁺ treatment correlates with a decline in levels of Proliferating Cell Nuclear Antigen (PCNA) (Figure 6B). Consistent with the FACS data, western analysis demonstrated that keratinocytes overexpressing either Np63 or Np63^p40 do not down-regulate PCNA levels in response to 0.12 mM Ca²⁺ (Figure 6B). The block in Ca²⁺-induced growth arrest also corresponds to a block in the induction of the cyclin dependent kinase inhibitor p21^WAF1. Whereas ß-gal control cultures exhibited an induction of p21^WAF1 under differentiating conditions, as previously described (27,30,41), both Np63 and Np63^p40 block Ca²⁺-triggered p21^WAF1 induction (Figure 6C). These results suggest that the N portion of the p63 protein blocks cell cycle regulation independent of the -terminus. This effect may be mediated at least in part via the regulation of p21^WAF1.

View larger version (36K): [in this window] [in a new window]   Fig. 6. Both Np63 isotypes block Ca2+ induced growth arrest. Keratinocytes were infected with adenovirus encoding Np63p40, TAp63, TAp63, Np63 or ß-gal. Seventeen hours post-infection cultures were either maintained in medium containing 0.05 mM Ca2+ or induced to differentiate by increasing extracellular [Ca2+] to 0.12 mM. (A) Twenty hours post-medium change, cultures were pulsed with 10 µM BrdU for the final 4 h prior to harvesting, and the S-phase population of each sample was determined using FACS analysis. At least three replicates were performed for each condition. Data are presented as mean ± SD, of a minimum of three independent experiments. (B) Protein lysates were prepared from cultures of primary keratinocytes overexpressing Np63, Np63p40 or ß-gal and maintained in medium containing 0.05 or 0.12 mM Ca2+ for the times indicated. Unlike ß-gal control cultures, the PCNA expression levels in keratinocytes overexpressing Np63 or Np63p40 remained elevated following culture in 0.12 mM Ca2+. (C) Keratinocytes overexpressing Np63, Np63p40 or ß-gal were harvested for western analysis following culture at 0.05 or 0.12 mM Ca2+ for 24 h. Induction of the cell cycle arrest protein p21WAF1 was observed in samples derived from control cultures of differentiating keratinocytes, but not in samples derived from cultures overexpressing Np63 or Np63p40.

 

	Discussion

Top Abstract Introduction Materials and methods Results Discussion Supplementary material References

 
That p63 is critical for development of normal squamous epithelium was demonstrated by the genetic deletion of all p63 isotypes in mice (42,43). Specific p63 isotypes are expressed during distinct stages of embryonic epidermal development (44) and roles attributed to these isotypes are beginning to be elucidated. Embryonic expression of TAp63 coincides with the onset of the epidermal stratification program and ectopic expression of TAp63 can initiate stratification of simple epithelium (44). Within the developed epidermis, Np63 has been postulated to maintain keratinocytes in a proliferative state (1,13,14). Consistent with these findings, chemically induced tumors of mouse skin are shown here to express high levels of Np63 (Figure 3). Additionally, we show that multiple p63 isotypes are expressed in normal primary neonatal keratinocytes and are individually modulated with in vitro differentiation. In addition to Np63, which has been regarded as the most abundant p63 isotype in keratinocytes, we observed a smaller Np63 variant, Np63_s, that migrates on an SDS–PAGE gel between Np63 and Np63^p40. Along with the protein results, quantification of N, and mRNA levels suggest that the Np63_s band reflects a novel p63 species or a post-translationally modified form of Np63^p40.

Previously we showed that overexpression of Np63 blocks keratinocyte differentiation, consistent with the decline in Np63 expression that normally occurs with differentiation. In contrast to Np63, Np63_s maintains robust expression in differentiated keratinocytes. To determine the potential contribution of an abbreviated Np63 form and the role of the N domain with minimal influence of C-terminal sequences, we evaluated the effects of introducing the truncated Np63 isotype, Np63^p40. Microarray analysis revealed potential functional differences with regard to differentiation between Np63 and Np63^p40 and this finding was confirmed by RT–PCR and western analysis. Unlike Np63, overexpression of Np63^p40 was compatible with keratinocyte differentiation (Figures 4A and 5). Similarly, Np63 harboring a mutation in the SAM domain is aberrantly expressed suprabasally in cells co-expressing markers of terminal differentiation (45). It has been previously shown that Np63 blocks differentiation-associated induction of the cdk inhibitor p21^WAF1 (30), a critical step in cell cycle withdrawal (46). Like Np63, Np63^p40 blocks p21^WAF1 induction, demonstrating that this function of Np63 is not influenced by the -tail. These findings suggest that the N-terminus maintains proliferative capacity of keratinocytes in the presence of 0.12 mM Ca²⁺, and the C-terminal -terminus, within the context of the Np63 subclass of isotypes, modulates differentiation of keratinocytes. A summary of the biological effects of overexpressing individual isotypes is presented in Table II. Whether the effects of the -tail on differentiation can be attributed to the SAM domain or other C-terminal sequences remains to be determined.

View this table: [in this window] [in a new window]   Table II. Summary of effects on keratinocyte proliferation and differentiation observed following overexpression of individual p63 isotypes

 
That overexpression of Np63^p40 permits induction of differentiation markers in a population of proliferating cells is unusual, but not unprecedented. Replication of suprabasal keratinocytes expressing keratin 10 has been suggested to be an early alteration occurring during mouse skin carcinogenesis (47). In squamous papillomas loss of the epithelial growth factor (EGF) receptor allows for premature migration of proliferating keratinocytes into the suprabasal compartment and subsequent co-localization of BrdU incorporation and keratin 10 expression (48). Similarly, keratinocytes lacking Rb co-express keratins 5 and 10 and incorporate BrdU suprabasally (49). In addition, keratin 6, a marker of proliferation, is co-expressed with keratin 10 in psoriasis (50).

Taken together, the results reported here suggest that maintenance of the balance of p63 isotypes is critical for keratinocyte growth regulation and begin to tease apart the functions of the individual domains within the context of a biological system. The TA and Np63 isotypes have been shown to possess overlapping as well as unique functions (51,52) and are capable of regulating each other (53). Although it is generally perceived that the Np63 isotypes work in a dominant negative manner towards p53 and the TAp63 isotypes, several studies have shown that in the right cellular context Np63 isotypes harbor transcriptional activation capacity (14,54,55). This function is attributed to additional transcriptional activation domains within the molecule (54,55), which might be regulated by interaction with co-factors. It is unclear to what extent this activity might influence the biological response of keratinocytes to differentiation signals. Current studies are aimed at elucidating potential co-factors contributing to the specific ability of Np63 to block keratinocyte differentiation. The elucidation of unique domain functions should help to clarify the contribution of p63 overexpression in cancers and potentially lead to therapeutic approaches to modulate the growth and differentiation properties of tumors with altered expression of p53 family members.

	Supplementary material

Top Abstract Introduction Materials and methods Results Discussion Supplementary material References

 
Supplementary material is available at: http://carcin.oxfordjournals.org/

	Acknowledgments

 
We gratefully acknowledge Dr Anil Rustgi for Np63 cDNA, Dr Shuntaro Ikawa for murine TAp63 cDNA and Dr David Sidransky for adenovirus encoding Np63^p40. We thank Drs Stuart Yuspa and Henry Hennings for providing tumor samples, Drs Luowei Li and Adam Glick for helpful advice regarding FACS methods, Jesse Quintero for assistance with Real-Time PCR assays and Jamie Zellers for assistance with tumor staining. We are grateful to Drs Stuart Yuspa and Wen Jin Wu for critical reading of the manuscript. This research was supported in part by the Intramural Research Program of the NIH (Center for Cancer Research, National Cancer Institute).

Conflict of Interest Statement: None declared.

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Received January 28, 2005; revised June 28, 2005; accepted July 28, 2005.

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