Nucleic Acids Research Advance Access originally published online on August 21, 2009
Nucleic Acids Research 2009 37(18):6092-6104; doi:10.1093/nar/gkp674
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Nucleic Acids Research, 2009, Vol. 37, No. 18 6092-6104
© The Author 2009. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses?by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Genomics |
Identification and functional characterization of two new transcriptional variants of the human p63 gene
1Dipartimento di Biochimica e Biologia Molecolare "E. Quagliariello", Università degli Studi di Bari, via Orabona 4, 70126 Bari and 2Istituto di Tecnologie Biomediche, Consiglio Nazionale delle Ricerche, via Amendola 122/D, 70125 Bari, Italy
*To whom correspondence should be addressed. Tel: +39 080 5443588; Fax: +39 080 5443317; Email: graziano.pesole{at}biologia.uniba.it
Correspondence may also be addressed to Anna Maria D’Erchia. Tel: +39 080 5443304; Fax: +39 080 5443317; Email: annamaria.derchia{at}biologia.uniba.it
Received May 31, 2009. Revised July 2, 2009. Accepted July 30, 2009.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACCESSION NUMBERSSUPPLEMENTARY DATAFUNDINGREFERENCES |
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p63 belongs to a family of transcription factors, which, while demonstrating striking conservation of functional domains, regulate distinct biological functions. Its principal role is in the regulation of epithelial commitment, differentiation and maintenance programs, during embryogenesis and in adult tissues. The p63 gene has a complex transcriptional pattern, producing two subclasses of N-terminal isoforms (TA and
N) which are alternatively spliced at the C-terminus. Here, we report the identification of two new C-terminus p63 variants, we named p63 
and 
, that increase from 6 to 10 the number of the p63 isoforms. Expression analysis of all p63 variants demonstrates a tissue/cell-type-specific nature of p63 alternative transcript expression, probably related to their different cellular functions. We demonstrate that the new p63 variants as N isoforms are active as transcription factors as they have nuclear localization and can modulate the expression of p63 target genes. Moreover, we report that, like Np63
, Np63 and sustain cellular proliferation and that their expression decreases during keratinocyte differentiation, suggesting their involvement in this process. Taken together, our results demonstrate the existence of novel p63 proteins whose expression should be considered in future studies on the roles of p63 in the regulation of cellular functions. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACCESSION NUMBERSSUPPLEMENTARY DATAFUNDINGREFERENCES |
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p63 is a member of a family of transcription factors, also including the tumor suppressor p53 and p73, which show high level identity in their main functional domains: the transactivation domain (TA), the DNA-binding domain (DBD) and the oligomerization domain (OD).
The human p63 gene, like the p53 and p73 genes, produces multiple protein isoforms as a result of alternative promoter usage and alternative splicing events (1). The promoter upstream of exon 1 generates the class of the TAp63 isoforms containing the TA at the N-terminus, while an alternative promoter in intron 3 leads to the expression of the Np63 isoforms lacking the N-terminal TA domain, although it has been shown that the Np63 isoforms can act as regulators of transcription through different TAs present in the distinct N-terminus (2) and C-terminus regions (3). Within each subclass, C-terminal alternative splicing events confer additional complexity. To date, three variants, , β and 
, which incorporate different portions of the C-terminus, have been described. The proteins are the longest including all terminal exons and containing the C-terminal sterile alpha motif (SAM) domain—a protein–protein interaction domain, followed by an inhibitory domain (TI), which is able to auto-inhibit the transcriptional activity of the TA subclass isoforms (4). The β variants lack exon 13 and consequently the SAM and the TI domains. The variants lack the C-terminal exons 11, 12, 13, 14, but incorporate an additional sequence of unknown function from intron 10. Therefore, the p63 gene expresses at least six different p63 isoforms (TAp63 , β, and Np63 , β, ), with a complex array of similarities and differences in their structural domains and transcriptional activities.
Despite the structural conservation between members of the p53 family, they are not functionally redundant as p53-family transgenic knockout mice develop distinct phenotypes, indicating that each protein has specific biological functions. Several lines of evidence suggest that, while the main role of p53 is related to the inhibition of tumor progression, p73 and p63 appear to be more directly involved in development and differentiation (5–7).
Current data indicate that p63 is an essential mediator of embryonic development. p63–/– mice have no epidermis and other stratified epithelia and also show striking defects in limb development (6). TA and Np63 isoforms are expressed during distinct stages of embryonic epidermal development. TAp63 isoforms are the first p63 isoforms to be expressed during embryonic development and they are necessary for the commitment to epithelial stratification while simultaneously blocking differentiation program. Therefore, a shift towards Np63 isoforms during later stages would be required to counterbalance the activity of TAp63, thereby allowing cells to respond to terminal differentiation cues (8,9). In adults, the Np63 isoform is predominantly expressed in the basal layers of stratified epithelial tissues, suggesting that it may contribute to maintain the proliferative potential of basal cells necessary for the epithelial stratification (10,11). In human p63, germline mutations have been reported in patients with ectodermal dysplasia syndromes, showing varying degrees of craniofacial, limb, skin and hair defects which resemble the phenotype of p63–/– mice (12).
The role of p63 in tumorigenesis is controversial. Initially, p63 was hypothesized to function as oncosuppressor based on its homology to p53. Mutations in the p63 gene are quite rare in human cancers and the gene maps in a region of chromosome 3 frequently amplified in squamous cell carcinoma. In primary head and neck squamous cell tumors and in other squamous epithelial cancers, Np63 is over-expressed and it was suggested that this form is required to maintain a stem cell-like state, allowing continuous proliferation and promoting tumor growth (13). Recently, it has been reported that p63+/– mice show a predisposition towards squamous cell carcinoma and that loss of p63 can cooperate with loss of p53 in tumor development, as dual heterozygous p63+/– and p53+/– mice display higher tumor burden and metastasis, compared to p53+/– mice (14). On the contrary, another independent study reported that p63+/– mice show no evidence of enhanced tumor development and that dual heterozygous p63+/– and p53+/– mice display reduced tumor burden compared to p53+/– mice (15).
In order to investigate the expression of the p63 gene, we have applied a bioinformatics approach (16,17), which detects transcript variants through the multiple alignment of all available transcript and/or EST sequences of a gene to the corresponding genomic sequence. Here we describe the identification of two novel human p63 C-terminal variants, that we have named and ; we describe the in vivo validation of these new p63 variants, and present results regarding their functional characterization.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACCESSION NUMBERSSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Bioinformatics
Novel p63 variants were identified using ASPicDB (10) (http://www.caspur.it/ASPicDB/index.php), which provided access to reliable annotations of the alternative transcriptional pattern of all human genes. AspicDB was used to obtain sequence data of every transcript and protein isoform predicted, and also to identify intron/exon boundaries of the genomic sequence corresponding to each variant, essential for the design of isoform-specific primer sets. Sequence data were aligned using Clustalw2 (http://www.ebi.ac.uk/Tools/clustalw2/index.html).
Cell cultures, transfection and in vitro keratinocyte differentiation
H1299, MCF-7, 293 and HaCat cell lines were maintained in Dulbecco's modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum (FCS), L-glutamine (2 mM), penicillin (100 U/ml) and streptomycin (100 µg/ml) at 37°C, 5% CO2. Transfections were carried out with Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. Eight micrograms of either empty pcDNA3 vector or vector containing the different Np63 variants (, β, , and 
) were used for a 60 mm plate of H1299 and MCF-7 cells (90–95% confluency). Cells were cultured in DMEM plus serum without antibiotics for 24 h. Then the medium was replaced with fresh complete medium and the cells were cultured for additional 24 h.
Normal human epidermal Keratinocytes were isolated from neonatal foreskin specimens derived from a normal human Caucasian (PromoCell). Cells were maintained in StemlineTM Keratinocyte Medium II (Sigma) supplemented with 1x Keratinocyte medium supplement, BPE free, L-glutamine (2 mM), penicillin (100 U/ml) and streptomycin (100 µg/ml) at 37°C, 5% CO2. Keratinocytes were differentiated by adding CaCl2 (2 mM final concentration) and reducing the supplement, BPE free, to 0.1x for 24 and 48 h.
Cell proliferation assay by BrdU incorporation
MCF-7 cells were transfected with 100 ng of either empty pcDNA3 vector (control) or containing p53 or the different Np63 variants (, β, , and ). Seventy-two hours later, bromodeoxyuridine (BrdU) incorporation, after 3 h pulses was determined by using Cell Proliferation ELISA BrdU (Roche) as described by the manufacturer.
Reverse transcriptase polymerase chain reaction and quantitative RT–PCR analyses
Total RNA from HaCat, H1299, MCF-7, 293 and primary keratinocyte cell lines was extracted using RNeasy Plus Mini Kit (Qiagen) according to the manufacturer's instructions. Total RNA from human muscle and brain was purchased from commercial sources (Ambion). cDNAs were synthesized from 1 µg of total RNA using QuantiTect® Reverse Transcription kit (Qiagen).
For Reverse transcriptase polymerase chain reaction (RT–PCR) experiments, 1 µl of cDNA was used as template for amplifications carried out with specific primer sets. The sequences of all primers are available upon request. PCR was run in the exponential region to allow semi-quantitative comparisons among cDNAs developed from identical reactions.
For quantitative RT–PCR, 1 µl of cDNA was used as template in real-time PCR assays, performed in triplicate on ABI PRISM 7900HT (Applied Biosystems). Each p63 variant was analyzed using the QuantiTect SYBR Green PCR Master Mix (Qiagen) with validated specific primers pairs. The PCR conditions were designed as follows: hot start at 95°C for 15 min; 45 cycles of amplification (94°C for 15 s, 62°C for 30 s, 72°C for 30 s); dissociation curve step (95°C for 15 s, 60°C for 15 s, 95°C for 15 s). For the quantification of ada, fasn, redd1, bax, p21, k14 and k1 transcripts, real-time PCR assays were performed using the TaqMan Universal PCR Master Mix (Applied Biosystems) with specific TaqMan Gene Expression Assays (Applied Biosystems) as described by the manufacturer.
Results were analyzed with the SDS 2.2.1 software. The comparative threshold (Ct) method was used to determine the relative ratio of transcripts. The relative quantification (RQ) was expressed as 2–Ct, where Ct was calculated as the average Ct for target variant minus the average Ct of endogenous gapdh in each sample, and Ct was calculated as the Ct of the sample minus the Ct of the calibrator. The data shown are the average from at least three independent experiments.
Construction of recombinant vectors
The full-length coding region of the human Np63 and Np63 variants was amplified from HaCat cDNAs using the Platinum® Taq DNA Polymerase High Fidelity (Invitrogen) and cloned into SacII/ApaI sites of a pcDNA3 expression vector containing the laminin A 5'UTR downstream the CMV promoter. The resulting vectors were termed pcDNA3-Np63 and pcDNA3-Np63.
Western blot analysis
For protein analysis, cells were washed twice in cold PBS and then lysed in RIPA buffer [50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholat, 0.1% SDS, protease inhibitors cocktail tablets (Roche)] for 1 h on ice. Lysates were then cleared by centrifugation at 10 000 g for 15 min at 4°C, aliquoted and stored at –20°C. Sixty micrograms of the total proteins, in 4x SDS–PAGE sample buffer, were heated at 100°C for 5 min, separated on 4–12% SDS polyacrylamide gels and transferred to nitrocellulose membranes (Hybond-ECL, Amersham Biosciences). Membranes were then blocked for 1 h in a PBS solution containing 3% non-fat milk powder and 0.1% Tween-20, and then probed at room temperature with Abp63 (4A4, Santa Cruz) for 3 h, in 3% milk, 0.1% Tween-20 PBS. For the analysis of keratinocyte differentiation, membranes were probed also with the following primary antibodies: Abp53 (DO-1, Santa Cruz) for 3 h, Abp21 (Calbiochem) for 3 h, Ab-actin (Calbiochem) for 1 h. After two 10 min washes in 0.1% Tween-20 PBS and one 10 min wash in PBS, proteins were visualized using ECL western blotting detection reagents (Amersham Pharmacia Biotech).
Luciferase assays
H1299 cells were plated in 35 mm tissue culture dishes (8 x 104 cells/dish) 24 h before transfection. Each well was then co-transfected using TransIT-LT1 Transfection Reagent (Mirus Bio), according to the manufacturer's instructions, with either empty pcDNA3 vector or containing the different Np63 variants (150 ng), the reporter vector (1 µg) along with Renilla pRL-SV40 vector (Promega) (10 ng).
Thirty-six hours after transfection, H1299 were lysed in Passive Lysis buffer (Promega) and the luciferase assay was performed using the Dual Luciferase assay system (Promega), according to the manufacturer's instructions. Data were normalized to the Renilla reporter signal. The results reported represent the average of at least three independent experiments and are shown with the standard deviations.
Immunofluorescence
H1299 cells were plated on a glass coverslip polylisine treated in six-well plates (4 x 104 cells/well). Twenty-four hours later, cells were transfected using TransIT-LT1 Transfection Reagent (Mirus Bio), with either empty pcDNA3 vector or containing the different Np63 variants (1 µg). Thirty-six hours after transfection, cells were fixed and permeabilized for 30 min with cold 50% methanol–50% acetone and then incubated at room temperature for 90 min with the Abp63 (4A4, Santa Cruz) at 1 : 50 dilution in DMEM plus serum. For cells transfected with empty pcDNA3 vector, only DMEM plus serum was added. After extensive washing, cells were incubated for 1 h at room temperature in the dark with FITC-conjugated secondary Ab against mouse Ig (Jackson) in DMEM plus serum (1 : 100). Nuclei were stained with DAPI (0.2 µg/ml). Fluorescence was analyzed with Axioplan 2 Imaging microscope (Zeiss).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACCESSION NUMBERSSUPPLEMENTARY DATAFUNDINGREFERENCES |
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In silico identification of two novel human p63 variants
Through the application of ASPIC, an algorithm for the prediction of alternative transcripts (16,17), we investigated the transcriptional pattern of the human p63 gene. Among the p63 transcripts predicted by the software, which included known isoforms, we detected two previously unidentified C-terminus p63 variants. The first variant, herein referred to as
, derives from the skipping of exons 12 and 13; the second variant, referred to as , is generated by a premature transcriptional termination in intron 10, retaining the 5' portion of intron 10, which immediately presents a stop codon (Figure 1A). The nucleotide sequences of the Np63 and transcripts were deposited in GenBank, with the accession numbers GQ202690
[GenBank]
and GQ202689
[GenBank]
, respectively.
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The
and transcripts produce proteins with different C-terminal regions (Figure 1B). In particular, the proteins contain 53/101 residues of the second TA (TA2), followed by eight unique amino acids after the oligomerization domain (OD). The proteins are the shortest isoforms, lack the TA2 domain and terminate, after the OD, with 22 amino acids shared with , β and isoforms; the proteins also lack the TA2 domain, maintain 21 common residues and terminate with 38 unique amino acids. The alignment among the C-terminal regions of and p63 proteins and the known , β and forms is reported in Figure 1C.
In vivo identification of and p63 isoforms in keratinocytes
To confirm the in vivo expression of the new and p63 isoforms, we used two human keratinocyte cell lines, HaCat and Nhek, as it is known that N isoforms of p63 are highly expressed in these cells (1). We performed RT–PCR experiments and western blotting analysis in order to validate both the new p63 transcripts and proteins.
RT–PCR experiments were carried out on total RNA using primer pairs specifically designed for the amplification of each variant, as shown in Figure 2. Amplification with primers For-ex11 and Rev-ex14 produced a 385 bp amplicon corresponding to that expected for the new transcript as well as 624 and 530 bp amplicons expected for the and β transcripts, respectively (Figure 2A). To identify the transcripts we used primers For-ex7 and Rev-ex10' producing only one amplicon, whose size of about 620 bp corresponded to that expected for the transcripts (Figure 2B). All amplicons were subcloned into a PCR cloning vector and sequenced. Alignment of the and amplicon sequences to the p63 gene sequence confirmed the in silico prediction of the new p63 transcripts exactly (data not shown).
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Because the
Np63 isoforms are the most abundant class in keratinocytes, we also amplified and sequenced the entire coding region of Np63 and Np63 transcripts from HaCat and Nhek cDNAs. Analysis of sequences confirmed the presence of these new Np63 transcripts.
Next, we analyzed expression of Np63 proteins in human keratinocytes and in a human breast carcinoma cell line. Lysates from HaCat and MCF-7 cells were resolved on a 4–12% SDS/PAGE gel together with size standard controls for each Np63 isoform (Np63, β, , and ). These standards were prepared by transfecting H1299 cells (which don’t express detectable p63 protein) with pcDNA3 vectors expressing the cDNAs of each Np63 isoform. Western analysis was performed using a pan-p63 antibody that recognizes the core DBD of p63. As shown in Figure 3, after a long exposure, HaCat and MCF-7 cell lysates revealed two bands corresponding to the Np63 and isoforms in addition to the known Np63 proteins. Moreover, differential expression of the Np63 isoforms was observed. Indeed, in HaCat cells Np63, and present lower levels of expression than Np63β and in particular Np63, which is the predominant isoform, while in MCF-7 cells, Np63 seems to be the least expressed p63 isoform.
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Expression of p63 transcripts in normal tissues and cell lines
In order to quantify the relative amounts of all p63 transcripts in different human tissues and cell lines and to determine their tissue-specificity, we performed real time RT–PCR assays.
Since p63 transcripts differ only in the 3' end of their coding region, we were not able to construct primers/probe sets for each variant. For this reason, we used the SYBR Green chemistry, designing specific primer pairs, mapping in the limited different sequence, to distinctly amplify each p63 variant (, β, , and ). In addition, to distinguish between the TA and Np63 transcript subclasses, we designed primer pairs able to amplify only the 5' end of TA and N transcripts.
Total RNA from primary muscle, brain and keratinocyte samples, embryonic kidney 293, breast cancer MCF-7 and HaCat cell lines was analyzed. First, we compared the relative levels of the TA and N p63 transcripts in each sample, using TA transcripts as calibrators (Figure 4A). Among tissues studied, only primary keratinocytes express high levels of N transcripts (
200-fold more than TAs), while muscle and brain show no detectable levels of N transcripts. We could thus consider the TA class as the predominant one in these samples. Among cell lines, HaCat cells exhibit the highest levels of N transcripts (400-fold more than TAs); in the MCF-7 cells, N transcripts are 2-fold more expressed than TA, while no detectable levels of Np63 mRNAs were observed in 293 cell line.
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Next, we assessed the relative levels of each p63 variant (
, β, , and ) in each sample examined, using the variant, which is generally the most expressed variant, as calibrator (Figure 4B). The profile of the TA and N p63 transcripts in these samples allowed us to associate the expression values obtained for each variant, to either TA or N subclasses. Results obtained, indicated that the new and p63 variants are expressed in all samples examined and, overall, all p63 variants show differences in their expression pattern. In fact, among tissues, the variant is the most expressed in all samples, with the exception of muscle, in which the variant showed the highest expression level. Interestingly, we found that in muscle, apart from the and variants the variant is the most highly expressed, while β and variants are almost undetectable. In brain, we found that, after the variant, the variant is more highly expressed than the , β and variants, the latter being the least expressed in this tissue. Among cell lines, the variant, is also the most highly expressed in all samples. HaCat cells showed an expression pattern similar to that of primary keratinocytes, with , and variants relatively less expressed than and β variants. In 293 cells, and variants showed a higher expression level than β and variants, while in MCF-7 cells, after the β, the and variants showed higher expression levels than the variant, in accord with observed protein expression levels (see Figure 3).
Finally, we compared the relative levels of each p63 variant among the tissues and cell lines examined (Figure 5). Among tissues, we observed that , β and variants are more expressed in keratinocytes than in muscle and brain, while, interestingly, the and variants present the highest levels in muscle tissue (respectively, 8- and 4-fold more expressed than in keratinocytes). Very low levels of all p63 variants are present in brain respect to the other tissues (Figure 5A). Among cell lines, HaCat cells show much higher expression levels of all five variants than MCF-7 and especially 293 cells (>9-fold expression drop) (Figure 5B).
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Nuclear localization of
Np63 and Np63 isoformsFor the functional characterization of the new p63
and variants, we focused our attention on the N isoforms. Given that the p63 gene encodes transcription factors, we investigated whether Np63 and Np63 proteins show nuclear localization. The Np63 or Np63 complete coding region was cloned in the pcDNA3 expression vector and the recombinant vectors were transfected in H1299 cells which were immunostained using the p63 4A4 antibody and a FITC-conjugated secondary antibody. Empty pcDNA3 vector was used as negative control, while cells transfected with vectors expressing Np63, Np63β and Np63 were used as positive controls. The subcellular distribution of all Np63 isoforms was assessed by fluorescence microscopy. As shown in Figure 6, we found that the ectopic expression of Np63 and Np63 isoforms led to an exclusively nuclear localization of fluorescence, suggesting that these variants, showing nuclear localization, could act as transcription factors and modulate the expression of p63 target genes. Identical cellular localization was, as expected, observed for the Np63, Np63β and Np63 proteins, used as controls.
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Transactivation activity of
Np63 and Np63 isoforms towards some p53 family target genesIn order to demonstrate that
Np63 and Np63 proteins have transcriptional activity, we performed luciferase assays, to assess whether they are able to directly activate some p53 family responsive promoters ( fasn, ada, bax and p21) (18–21). To this end, different luciferase constructs—pGL3promoter-fasn, pGL3promoter-ada, pGL3basic-bax and pGL3basic-p21—were co-transfected into p53 null H1299 cells either with a pcDNA3 empty vector as control or with the pcDNA3 vectors expressing Np63 and Np63 and the known Np63 isoforms (, β, ), to compare the activity of all Np63 isoforms. In parallel, for each assay, the intracellular level of transfected proteins was assessed by western blot.
As shown in Figure 7A, we found that Np63 and Np63 have significant transcriptional activity towards fasn RE, with 7- and 6-fold activation, respectively, compared with the control, although Np63 and β are the strongest transactivating variants on this element, with 21- and 18-fold activation of the target, respectively. The remaining variant, Np63, shows similar levels of target activation to Np63. Considering that Np63β and show the lowest expression levels, the transactivation activity of these two isoforms could be even greater than that evaluated.
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Regarding the ada RE (Figure 7B), we found that
Np63 and show transactivation activity similar to that of Np63 and β, with an increase of the luciferase activity of about 7-fold for Np63 and of about 5.5-fold for Np63, compared with the control. Weaker transactivation activity is showed by Np63, which induced only 3-fold activation of the target. As Np63 and Np63 transfected proteins have higher expression levels than other isoforms, their transactivation activity could be slightly lower than that estimated.
The activation of the bax RE is also differentially modulated by the Np63 isoforms (Figure 7C). We found that Np63 and Np63 activate the bax RE, with an increase of the luciferase activity of about 8-fold for Np63 and of about 10-fold for Np63, like Np63, while Np63β and Np63 transactivate this element more efficiently, with an increase of about 53-fold and 39-fold, respectively, with respect to the control.
Finally, Figure 7D shows the transcriptional activity of the Np63 isoforms on the p21 RE. Np63 and Np63, do not show relevant transcriptional activity on the p21 promoter, as already observed for Np63 (2). In agreement with other groups (2,3), we found that Np63β and Np63 are able to activate the p21 RE, with an increase of about 9- and 4-fold activation of the target, respectively.
Taken together, these results indicated that the new Np63 isoforms, Np63 and Np63, show a significant transactivation activity towards the p63 REs examined, except for p21 and that the Np63 isoforms differ in their regulatory activity on the p63 REs.
Effect of Np63 and Np63 isoforms over-expression on p63 target gene expression and cellular proliferation
To determine whether the new Np63 variants are able to modulate the endogenous expression of p63 target genes, we performed quantitative RT–PCR to detect the mRNA levels of some Np63 positive (fasn, ada, redd1 and p21) and negative (bax, C/EBPdelta and igfbp-3) genes (18–24) in the presence of over-expression of the five Np63 isoforms in H1299 and MCF-7 cell lines (Figure 8A and B). The levels of exogenously expressed Np63 isoforms in H1299 and MCF-7 cells were monitored by western blot analysis (Figure S1). We found that, in both cell lines, fasn, ada and redd1 genes appear to be upregulated, to varying degrees, by Np63 and Np63 as well as by the other isoforms (with the exception of redd1 which is downregulated by Np63 in H1299 cells). In particular, in H1299 cells, Np63 increases the levels of fasn mRNA more than the other isoforms, and in MCF-7 cells Np63 determines the highest increase of ada mRNA levels. The p21 expression is upregulated by Np63β and slightly by Np63 and Np63 in H1299 cells, in accordance with luciferase assay results, while no p21 activation by all isoforms is detected in MCF-7, as previously reported for Np63 and Np63 (2). In both cell lines, bax expression is downregulated by all Np63 isoforms, suggesting that, in conditions of over-expression, the Np63 isoforms repress the expression of the pro-apoptotic bax gene in accordance with their known antiapoptotic role (25). This result is in contrast with the luciferase results, but could be due to the artificial nature of the luciferase construct. In both cell lines, the other two negative target genes C/EBPdelta and igfbp-3 are downregulated by Np63 and Np63 while, interestingly, igfbp-3 appears to be upregulated by Np63β. These results demonstrate that Np63 and Np63, like the other Np63 isoforms, are able to modulate in vivo the expression of Np63 target genes and that this modulation varies in the two cellular contexts.
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On the basis of previous data and in order to assess whether
Np63 isoforms affect cellular proliferation, we compared MCF-7 cells over-expressing Np63 isoforms with wild-type cells, by BrdU incorporation (Figure 8C). The levels of exogenously expressed Np63 isoforms in MCF7 cell line were monitored by western blot analysis (data not shown). Interestingly, the ectopic expression of the Np63, Np63 and isoforms, but not of p53 and Np63β and isoforms, increased cell proliferation rate to different degrees: Np63 showing a higher efficacy than Np63 and . These data demonstrate that Np63 and isoforms, like Np63, have effects distinct from those of Np63β, Np63 and p53 on the cell cycle and that their over-expression increases cell proliferation rate.
Expression of Np63 and Np63 isoforms during keratinocyte differentiation
It has been reported that Np63 expression is modulated during keratinocyte differentiation; being required to maintain the proliferative potential of basal cells of the epidermis but being down-regulated in terminally differentiated cells in more luminal strata (8). We investigated the expression of the new Np63 and Np63 isoforms during keratinocyte differentiation, by analyzing the expression pattern of all Np63 isoforms in differentiating keratinocyte cells, used as a model of squamous epithelium. To induce differentiation of proliferating keratinocytes, we altered the extracellular Ca2+ concentration from 0 mM to 2 mM and the medium serum concentration from 1x to 0.1x. The expression of the Np63 isoforms was analyzed at 24 and 48 h following the differentiation stimulus.
RT–PCR experiments (Figure 9A), performed using primer pair sets able to amplify the full-length Np63 transcripts, and qRT–PCR analysis (Figure 9B) of Np63 variants showed a progressive reduction of the levels of all Np63 transcripts, in particular, at 48 h after the differentiation stimulus. Figure 9C shows the Np63 protein pattern during the differentiation process which is similar to that of the transcripts, as a reduction of all Np63 isoforms is observed, such that, only Np63 remains detectable 48 h after the differentiation stimulus.
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As controls for the differentiation induction, we first monitored the levels of p21 and p53 proteins, involved in cell cycle block. As shown in Figure 9D, at 24 and 48 h after the differentiation stimulus, p21 levels and especially p53 levels increased, with respect to proliferating cells.
Moreover, we analyzed the transcript levels of keratin 14 and keratin 1, two keratinocyte differentiation markers, to confirm the proper differentiation status of Nhek cells (Figure 9E). We found a remarkable progressive increase of keratin 1 mRNA and a decrease, more evident at 48 h, of the transcript of keratin 14, a marker of the less differentiated layers of epidermis, which is also positively regulated by Np63 (26). In addition, we examined the expression of other Np63-regulated genes (ada, redd1, keratin 4) (Figure 9E). Consistent with the reduction of all Np63 isoforms during differentiation, we also observed a relative decrease of transcripts of ada and redd1 target genes and an increase of keratin 4 expression, which was reported to be downregulated by Np63 (27).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACCESSION NUMBERSSUPPLEMENTARY DATAFUNDINGREFERENCES |
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The p63 gene, along with p53 and p73, belongs to the p53 gene family. Although their products show some common structural and functional features, each protein seems to have specific biological functions. In fact, while p53-deficient mice grow normally but undergo spontaneous tumor development, p73 and p63 knockout mice exhibit severe developmental and differentiation defects (6,7,28). A common feature of the three genes is to produce a great variety of isoforms, either derived from the use of an internal cryptic promoter or by alternative splicing, giving rise to a network of proteins whose fine modulation is fundamental in the regulation of biological activities, ranging from development and differentiation to growth arrest and apoptosis (29). The p63 gene generates the expression of two subclasses of isoforms, namely, those containing the TA, called TA isoforms, and those lacking this domain, called
N isoforms. Moreover, three alternative splicing events have been identified, producing the , β and , variants, which incorporate various portions of the C-terminus, for both TA and Np63 isoforms.
Therefore, until now six different p63 isoforms (TAp63 , β, and Np63 , β, ) have been identified, with a complex array of similarities and differences in their structural domains and transcriptional activities.
By using the ASPIC algorithm (16,17), a tool for alternative splicing prediction, based on multiple transcripts and/or EST sequence comparison and alignment against the gene sequence, we identified two new human p63 C-terminus variants, one we named , deriving from an alternative splicing event between exons 11 and 14, and the other, we defined , that keeps the 5' portion of intron 10, which immediately presents a stop codon. Transcript isoforms homologous to the and variants were not detected for p73 gene. The ASPIC algorithm, unlike other alternative splicing prediction methods which perform independent single transcript alignments with the genomic region, carries out an optimized alignment of all the gene-related transcripts with the aim of minimizing the number of predicted splice sites and consequently of the relevant transcript variants. The reliability of the ASPIC algorithm along with the significant number of EST sequences supporting these new p63 variants, prompted us to investigate the presence of these new p63 proteins in vivo.
Here, first we identified these new p63 variants in keratinocytes and MCF-7 cells, indicating that the ASPIC algorithm could be considered to provide valid starting point in the search for transcriptional variants of genes of interest (Figures 2 and 3). In keratinocytes, known to express high levels of p63 as N variants, we observed a differential expression of the novel variants with respect to the other known isoforms. In particular, Np63 is highly expressed respect to Np63 and Np63 and, as expected, the N and β variants are the predominant isoforms (Figure 4B).
We extended the study of the expression of all five p63 variants, by qRT–PCR methodology, to different normal human tissues and cell lines in order to determine whether it was tissue-specific (Figure 4). The results confirmed that the p63 variants are differentially expressed, either as TA and N form, in tested samples and that the two new variants, and , show interesting expression profiles. In brain, where only the TA class was detected, the variant is the most expressed after the variant, and in muscle tissue, the TAp63 transcript shows levels only slightly lower than TAp63. Among the cell lines analyzed, HaCat cells show a p63 expression profile similar to keratinocytes, while in MCF-7 cells, variants show similar expression level to β and variants, while variants show the lowest expression. Interestingly, in 293 cells, the and variants seem to be the predominant variants after . We also compared the relative levels of each p63 variant among the tissues and cell lines (Figure 5), showing that , β and variants are more expressed in keratinocytes, while and variants are present at higher levels in muscle tissue. Very low levels of all p63 variants are present in brain with respect to the other tissues analyzed. Among cell lines, very low expression of all variants were observed in MCF-7 and 293 cells with respect to HaCat cells.
After demonstrating the in vivo expression of the new and p63 variants, we have performed initial functional characterization. An indication that, like other isoforms, Np63 and Np63 act as transcription factors, was derived from the study of their cellular localization and transactivation activity. In fact, both Np63 and Np63 show a nuclear localization (Figure 6), have significant transactivation activity towards the p63 REs examined (Figure 7) and are able to regulate in vivo the expression of Np63 positive and negative target genes although with different efficiency with respect to the other Np63 isoforms (Figure 8A and B). The effect of the ectopic expression of the Np63 isoforms on the expression of the target genes is variable in the two cellular contexts examined, suggesting that other cell-specific transcription factors may participate with Np63 proteins in the regulation of these genes. Moreover, we show that the ectopic expression of the Np63 and isoforms, like Np63, increase the cell proliferation rate, while Np63β, Np63 and p53 reduce proliferation rates (Figure 8C). Taken together, these data may suggest that Np63 and isoforms, as Np63, could have a role in sustaining cell proliferation as they activate genes involved in cell cycle progression (fasn and ada) but not the pro-apoptotic gene bax.
The and p63 proteins lack one of the two TAs (TA2), which was demonstrated to be required for the transcriptional activity of the N isoforms (3). Overall, our results demonstrate that the N forms of these variants are functionally active, suggesting that the TA (TA1*) (2) present at the N-terminus of all N isoforms contributes significantly to their transcriptional activity.
Moreover, we studied the expression profile of the new Np63 variants during keratinocyte differentiation. Many studies report a role for p63 in the differentiation of epidermis, a key process by which the epithelial cells acquire their proper morphological and functional properties (30). However, it remains unclear how exactly this takes place. The basal compartment of stratified epithelia is made of cells with high proliferative capacity that replenish the terminally differentiated populations in the more luminal strata. Np63 is the isoform predominantly expressed in this basal compartment while TA isoforms are not detectable. It is suggested that Np63 is required to maintain the proliferative potential of basal cells and to allow the initial induction of keratinocyte differentiation, while it must be downregulated to allow terminal differentiation (9).
Our results confirm that the differentiation process is associated with lower levels of all Np63 isoforms (Figure 9) not only Np63, at both the transcripts and protein levels, although their relative abundance seems to be maintained. Moreover, during differentiation, we observed a reduction of the expression of some known Np63 target genes, consistent with the reduction of the Np63 variants, supporting the hypothesis that all isoforms, including Np63 and Np63, could, along with Np63, play a pro-proliferative role in this physiological condition. Further studies to define the signals controlling the balance in the expression of all the p63 isoforms will help to better understand the contribution of p63 to proper epidermal homeostasis.
Taken together, our results demonstrate the existence of new p63 variants, which are functionally active as transcription factors, increasing to 10 the number of the isoforms that are produced by the p63 gene.
The implications of this study open interesting avenues for future investigation. In particular, we have demonstrated tissue/cell type-specific expression of p63 alternative transcripts, suggesting possible relationships to their different cellular function. A better understanding of the regulation of expression of all p63 possible variants will thus be essential for meaningful p63 functional studies. Indeed, future studies on p63 function should take into account the possible presence of these new variants in the tissue or cell type examined, which if ignored would provide partial and/or potentially incorrect results.
Furthermore, the existence of new p63 variants increases the known complexity of the crosstalk among all the p53 family members and of their mutual regulation in the maintenance of the proper cellular growth of epithelial tissues.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACCESSION NUMBERSSUPPLEMENTARY DATAFUNDINGREFERENCES |
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GQ202690 and GQ202689.
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Supplementary Data are available at NAR Online.
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Telethon (grant number GGP06158), Progetto Strategico Regione Puglia and Grant 2008 of the Bari University (ex 60%). Funding for open access charge: Associazione Italiana Ricerca sul Cancro (AIRC).
Conflict of interest statement. None declared.
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The authors thank Dr D. Horner for suggestions and critical reading of the manuscript.
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