Introduction

The transcriptional activator p63 is a p53 paralog that is expressed in basal cells and somatic stem cells of stratified epithelia, in myoepithelial cells of the breast, salivary glands, and in the proliferative compartment of gastric mucosa (Yang et al., 1998, 1999). Owing to the use of two promoters and complex alternative splicing, p63 encodes six isoforms (Yang et al., 1998). Of these, TA isoforms structurally resemble p53 and contain an N-terminal transactivation domain, whereas Delta-N (N) isoforms lack this transactivation domain (Yang et al., 1998). Dominantly inherited mutations in the p63 gene are found in a number of human ectodermal dysplasias, including ectrodactyly ectodermal dysplasia-cleft lip/palate (EEC) syndrome, limb-mammary syndrome (LMS), ankyloblepharon ectodermal dysplasia clefting (AEC) and non-syndromic split-hand/split-foot malformation (SHFM) (Rinne et al., 2006). Genotype–phenotype correlations exist in that mutations causing EEC syndrome, for example, are not found in AEC, LMS, or SHFM (van Bokhoven and Brunner, 2002). Moreover, there is a clear correlation between the position where the mutation occurs and the observed syndrome. The majority of mutations found in the EEC syndrome are missense mutations generating amino acid substitutions in residues predicted to contact DNA (Rinne et al., 2006). As the DNA binding domain is present in all p63 isoforms, all isoforms of p63 are affected by these mutations. Mutations in exon 13 and exon 14, affecting only the -isoforms of p63, are almost exclusively associated with AEC (McGrath et al., 2001; Rinne et al., 2006). DNA-binding mutants often act as dominant-negative molecules, although whether this is also the case for mutant p63 proteins remains to be determined.

Mice lacking p63 die soon after birth with several developmental defects, particularly in limb and skin development (Mills et al., 1999; Yang et al., 1999). Defects in limb morphogenesis in p63 null mice were evident as early as embryonic day 9.5 (E9.5). In wild-type (wt) mice, during this interval, p63 is expressed in the surface ectoderm as well as in the ectoderm covering the limb buds and branchial arches. Although still a matter of debate (Laurikkala et al., 2006; Suh et al., 2006), it has been found that TAp63 isoforms, the first p63 isoforms expressed during epidermal development, are required for the commitment to stratification while they inhibit terminal differentiation (Koster and Roop, 2004; Koster et al., 2004). After commitment to stratification has occurred, Np63 isoforms induce the expression of genes that are required for later stages of epidermal morphogenesis (Koster et al., 2007).

Interestingly, I-kappaB kinase alpha (IKK)-deficient mice display developmental defects, including skin, craniofacial, and limb defects, showing some similarities with p63 null mice (Hu et al., 1999; Li et al., 1999; Mills et al., 1999; Takeda et al., 1999; Yang et al., 1999). Although IKK is a protein kinase that is part of the IKK complex, its role in epidermal differentiation is independent of its kinase activity or NF-B activation (Hu et al., 1999). Interestingly, expression of IKK in the epidermis is required not only for epidermal development but also for the development of tissues derived from the mesoderm and neural crest (limbs and craniofacial structures) (Sil et al., 2004) and to switch on the differentiative program by favoring keratinocyte cell cycle arrest (Descargues et al., 2008). A recent study (Candi et al., 2006) has identified IKK as a transcriptional target for p63 with TAp63 being a better activator than Np63 in transcriptional assays. In our previous work (Koster et al., 2007), we showed that epidermal-specific Np63 downregulation in mice leads to increased keratinocyte proliferation, which correlates with reduced expression of IKK. Furthermore, these mice display a skin phenotype characterized by skin fragility that may resemble that of AEC patients (Koster et al., 2007). We thus hypothesized that IKK and p63 may genetically also interact in human cells, with IKK functioning downstream of p63. Furthermore, we hypothesized that mutant p63 proteins expressed in ectodermal dysplasia patients may interfere with the induction of IKK expression and that the alteration of this pathway may contribute to the phenotype observed in ectodermal dysplasia patients. In this study, we show that p63 directly induces IKK transcription and that mutant p63 proteins expressed in ectodermal dysplasia patients interfere with IKK induction.

Results

IKK is a direct transcriptional target of p63 in keratinocytes

IKK promoter contains at least three potential p63 responsive elements (Osada et al., 2005a, 2005b; Candi et al., 2006). To determine whether p63 could induce IKK transcription, we generated a luciferase reporter construct (IKK-luc) containing a 1.2 kb fragment of the IKK promoter and performed transient transactivation assays. Exogenously expressed Np63, Np63, Np63, and TAp63 strongly induced the IKK reporter construct, whereas TAp63 and TAp63 weakly induced this reporter (Figure 1a). We also observed activation of the IKK promoter by exogenously expressed Np73 and TAp73 (Figure 1a). In contrast, p53 expression did not induce transcription from the IKK promoter either in U2OS cells (Figure 1a) or in the immortalized keratinocyte cell line HaCaT (data not shown). Although this is in disagreement with the previously described role of p53 as a transcriptional repressor of IKK in an acute lymphoblastic leukemia cell line (Gu et al., 2004), this discrepancy could potentially be explained by cell-type specificity.

Figure 1.

p63 activates IKK transcription. (a) p63 activates IKK reporter gene. U2OS cells were transfected with IKK reporter plasmid (0.1 g), pRL-null plasmid (0.02 g), and expression vectors for different p63 and p73 isoforms or p53 (1.5 g). After 24 hours, cells were harvested and luciferase activity was determined (left panel). Protein expression was evaluated by western blot analysis with anti-Myc, anti-HA, and anti-p53 antibody (Ab) (right panel). (b) Exogenously expressed p63 and p73 isoforms induce IKK mRNA and protein expressions. U2OS cells were transfected with 2 g of the indicated plasmid in 35 mm dishes. Cells were harvested 24 hours after transfection and total mRNA was extracted and used for RT-qPCR. Results are expressed as IKK expression relative to ARP (human acidic ribosomal protein P0) expression (left panel). In a parallel experiment, U2OS cells were transfected with 2 g of the indicated plasmid in 35 mm dishes. Cells were harvested 24 hours after transfection, and total cell lysates (25 g) were subjected to immunoblot analysis with an anti-IKK Ab to determine IKK expression. Actin expression was determined as a loading control. Levels of expression relative to pCDNA (Ctr)-transfected cells are indicated (right panel). (c) p63 and p73 directly interact with the IKK promoter. U2OS cells were transfected with the indicated plasmids. Cells were subjected to ChIP, and the recovered chromatin was amplified with IKK promoter-specific primers and normalized to protein expression (Figure S1). The ChIP assay was repeated on primary human keratinocytes under proliferating or differentiating conditions using anti-p63-specific polyclonal Abs (right panel).

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The ability of p63 to induce endogenous IKK expression was tested in transient expression experiments in U2OS cells. As shown in Figure 1b, exogenously expressed Np63 and Np73 isoforms induced an increase in the levels of endogenous IKK mRNA (left panel) and protein (right panel) better than the TA isoforms did. Expression levels of different p63 and p73 isoforms are shown in Figure S1A.

To determine if p63 isoforms directly interact with the IKK promoter, we carried out chromatin immunoprecipitation (ChIP) experiments after transient transfection of U2OS cells with myc-tagged p63 expression plasmids. Cross-linked chromatin was immunoprecipitated with specific antibodies (Abs), and the presence of recovered IKK promoter fragments was analyzed by PCR using primers specific for the proximal region of the putative IKK promoter. Quantification of p63-bound IKK promoter fragments after normalization to the amount of transfected p63 protein (Figure S1B) indicated that Np63, TAp63, and Np63 had a higher affinity for the IKK promoter than TAp63 did (Figure 1c, left panel). The ChIP assay was repeated in primary human keratinocytes placed under proliferating or differentiating conditions (Figure 1c, right panel). We observed a transient increase in p63 binding to IKK promoter peaking 8 hours after differentiation stimulus (Figure 1c, right panel).

These data indicate that p63 isoforms can induce IKK transcription, acting most likely through direct binding to its promoter.

Np63 induces IKK during keratinocyte differentiation

To study the regulation of IKK transcription during keratinocyte differentiation, we examined IKK mRNA levels by real-time quantitative PCR (RT-qPCR) in normal human epidermal keratinocytes (NHEK) subjected to a differentiation stimulus (2 mM CaCl₂). IKK mRNA levels increased at early times (8 and 24 hours) after the induction of NHEK differentiation (Figure 2a, left panel). As expected, Np63 mRNA levels decreased upon differentiation (Figure 2a, right panel), suggesting that p63 may be required to induce IKK transcription but not to maintain it. The early induction of IKK transcription upon keratinocyte differentiation correlates with high levels of IKK protein expression in the nuclei of basal and suprabasal keratinocytes in normal human skin (Figure 2b, upper panels) and colocalization of IKK with p63 in most cells of the basal and suprabasal layers (Figure 2b, lower panels). To determine whether the subcellular localization of IKK was also affected by differentiation of human keratinocytes, we performed immunoblotting using an anti-IKK Ab on cytoplasmic and nuclear extracts of normal human keratinocytes. We observed an increase in the nuclear fraction of IKK protein upon keratinocyte differentiation (Figure 2c), confirming previous observations obtained in mouse keratinocytes (Sil et al., 2004; Descargues et al., 2008).

Figure 2.

Nuclear IKK expression increases at the onset of keratinocyte terminal differentiation. (a) IKK expression increases at the onset of keratinocyte differentiation. Human primary keratinocytes were treated with 2 mM CaCl₂ for 8 or 24 hours. Relative mRNA abundance was determined by RT-qPCR for IKK, K1 (left panel), or Np63 (right panel). (b) p63 and IKK colocalize in keratinocytes in human epidermis. Immunohistochemical staining of normal skin sections was performed with a monoclonal anti-IKK antibody (Ab) (see the Materials and Methods section), and upper panels represent original magnifications of 10 and 40 acquisition of the same section. Analysis of IKK and p63 expressions in the human epidermis was performed by confocal microscopy after immunofluorescence staining (scale bar=20 m). (c) IKK nuclear translocation is induced by CaCl₂ treatment. Human primary keratinocytes were treated with 2 mM CaCl₂. Cells were harvested 24 hours after treatment, and nuclear/cytoplasmic extracts were analyzed by western blot analysis using an anti-IKK Ab. Actin, -tubulin, and -tubulin expressions were used as a loading control to determine the purity of cytoplasmic extracts.

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To determine whether Np63 proteins are involved in IKK induction, we used small interfering RNAs (siRNAs) to selectively downregulate Np63 transcripts in primary keratinocytes. The specificity of siRNAs was determined by transient transfection experiments performed in U2OS cells in which we co-transfected expression plasmids encoding for Np63 together with control siRNAs (GFPsiRNA), Np63-specific, or TAp63-specific siRNAs. We observed a high efficiency of Np63-specific siRNA in downregulating Np63 protein expression (Figure 3a, left panel). The same siRNAs were used to downregulate endogenous Np63 in primary human keratinocytes (Figure 3a and b) or in primary mouse keratinocytes (Figure S2). The transfection of Np63 siRNAs resulted in a marked downregulation of Np63 expression in both proliferating and differentiating keratinocytes. Downregulation of Np63 had no effect on the basal levels of IKK protein, whereas calcium-induced levels of IKK were significantly decreased in Np63-silenced cells (Figure 3a, right panel). Np63 downregulation also decreased the expression of K1 early differentiation marker (Figure 3a, right panel). These results were confirmed by evaluating the levels of IKK mRNA in keratinocytes in which Np63 was downregulated (Figure 3b), and suggest that Np63 is involved in the induction of IKK at early phases of keratinocyte differentiation. Indeed, the downregulation of Np63 also resulted in a failure to induce early differentiation markers of keratinocytes such as K1 (Figure 3b, right panel), indicating that Np63 controls the formation of suprabasal layers. We also confirmed these results in primary mouse keratinocytes (Figure S2). In addition, as Np63 expression levels are reduced in the suprabasal layers of the epidermis (Yang et al., 1998), these data are consistent with the existence of Np63-independent mechanisms for maintaining IKK expression in terminally differentiated cells.

Figure 3.

Np63 is required for induction of IKK expression by differentiation. (a) Specificity of Np63 siRNAs. U2OS cells were co-transfected with 1 g of Myc-tagged Np63 expression plasmids, and the indicated siRNAs and cell lysates were subjected to immunoblotting with anti-myc or anti-actin antibodies (left panel); primary human keratinocytes were transfected with specific siRNAs and then kept under proliferating or differentiating (2 mM CaCl₂) conditions. After 24 hours, cells were harvested and 25 g of total lysates were subjected to immunoblot analysis to verify p63, IKK, K1, or actin expression levels (right panel). Expression levels relative to control (first lane) were quantified. (b) The downregulation of Np63 isoforms abrogates CaCl₂-induced activation of IKK transcription. Primary human keratinocytes maintained under proliferating or differentiating (2 mM CaCl₂) conditions were transiently transfected with specific siRNAs. Relative mRNA abundance was determined by RT-qPCR for IKK (left panel) and K1 (right panel). (c) Reintroduction of IKK in Np63-deficient keratinocytes restores calcium-induced exit from the cell cycle. Primary keratinocytes were transfected with indicated siRNAs. Sixteen hours after transfection, cells were infected with Ad-IKK. The next day, the cells were induced to differentiate by the addition of calcium (2 mM). BrdU was then added for 6 hours and its incorporation measured by a specific kit. Results are expressed as percentage of positive cells and error bars represent SD. Results are representative of three independent experiments.

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In addition to reduced levels of K1 differentiation markers, Np63-downregulated cells also display increased proliferation as determined by BrdU incorporation assay (Figure 3c). This is consistent with the observation made on the Np63-knockdown mice skin (Koster et al., 2007) and with the phenotype of AEC skin (Figure 5c). Given the role of IKK as a critical regulator of cell cycle exit in keratinocytes (Koster et al., 2007; Descargues et al., 2008), we attempted to rescue the correct proliferation rate by reintroducing IKK in Np63-deficient keratinocytes. To this aim, human keratinocytes were transfected with control or Np63-specific siRNAs and then infected with mock or IKK-expressing adenoviruses and placed under differentiation conditions. Twenty-four hours after infection, BrdU assay was performed to determine the proliferation rate. As shown in Figure 3c, the reintroduction of IKK in keratinocytes restored low proliferation rate. These data suggest that IKK may act as the Np63 target gene required for correct exit from the cell cycle upon differentiation stimulus, a step necessary to achieve terminal differentiation.

Figure 5.

Dominant-negative activity of p63 mutants on Np63-induced IKK expression. (a) TE1 cells were transfected with wild-type Np63 alone or in combination with the indicated expression plasmids. After 24 hours, cells were harvested and the IKK mRNA expression normalized to ARP levels was determined by RT-qPCR. (b) TE1 cells were transfected with wild-type Np63 alone or in combination with the indicated expression plasmids. Cells were harvested after 36 hours, and total cell lysate was immunoblotted with specific antibodies (Abs). The relative amount of endogenous IKK protein was quantified by densitometric analysis. (c) Skin sections from healthy donors (upper panels) and from the lesional skin of a patient affected by AEC syndrome (I537T mutation in the SAM domain of p63) (lower panels) were subjected to immunohistochemistry with specific Abs to reveal the pattern of expression of IKK (left panels), K1 (middle panels), and the proliferation marker Ki-67 (right panels). Scale bar=20 m. Total RNA extracted from normal skin sections and from AEC skin sections was subjected to RT-qPCR to determine IKK mRNA expression levels (right histogram). Bars represent the mean expression (SD) obtained from three different sections of normal subjects and of the AEC patient.

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p63 mutant proteins expressed in ectodermal dysplasia patients influence IKK transcription

Given that some defects observed in ectodermal dysplasias caused by p63 mutations are also present in IKK knockout mice, we hypothesized that mutant p63 proteins expressed in ectodermal dysplasia patients may have a reduced ability to induce IKK. To test this hypothesis, we determined the ability of mutant Np63 proteins to activate the IKK reporter gene and to induce endogenous IKK expression. To this aim, we used expression plasmids expressing different p63 mutants found in ectodermal dysplasias including DNA binding domain mutants (Np63-306) and truncation mutants (Np63-fs) of EEC syndrome, Np63-518 and Np63-536 SAM domain mutants of AEC syndrome, Np63-DAA of LMS, and Np63-639 of SHFM (Figure 4a). We initially performed transcriptional assays in the epithelial cell line TE1 (North et al., 2002) and observed that mutant Np63 proteins expressed in EEC and AEC patients (Np63-EEC and Np63-AEC, respectively) displayed a reduced ability to activate the IKK reporter gene (Figure 4b). In particular, Np63-AEC proteins had a reduced ability to activate the IKK reporter construct. Similarly, mutant Np63-EEC proteins showed a reduced ability to activate the IKK reporter construct. Mutant Np63 proteins expressed in LMS and SHFM patients (Np63-LMS and Np63-SHFM, respectively), on the contrary, did not show significant differences in their ability to activate the IKK reporter (Figure 4b).

Figure 4.

Transcriptional regulation of the IKK promoter by Np63 mutants expressed in ectodermal dysplasias. Transcriptional regulation of the IKK promoter by Np63 mutants expressed in ectodermal dysplasias. (a) Expression levels of myc-tagged wt Np63 and Np63 mutants L518V, Q536L, FS, C306R, 1743 DelAA, and E639X were analyzed in TE1 cells by immunoblot with an anti-myc antibody. (b) TE1 cells were co-transfected with the IKK reporter plasmid (0.1 g), pRL-null plasmid (0.02 g), and expression vectors for Np63 wt and mutants L518V, Q536L, FS, C306R, 1743 DelAA, and E639X (1.5 g). After 24 hours, cells were harvested and luciferase activity was determined. (c) TE1 cells were transfected with the indicated plasmids and subjected to total mRNA extraction. IKK mRNA expression was determined by RT-qPCR after normalization to ARP expression.

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The analysis of IKK mRNA transcript levels in TE1 cells transfected with Np63-AEC or Np63-EEC constructs confirmed the defective ability of Np63-AEC and Np63-EEC to induce IKK transcription and showed reduced ability of the LMS mutant to induce accumulation of IKK mRNA (Figure 4c). Similar results were obtained in the U2OS cell line (data not shown).

p63 mutations that cause ectodermal dysplasias are heterozygous, and mutant p63 proteins expressed in ectodermal dysplasia patients may have a dominant-negative effect toward wt p63 on the activation of p63 target genes. To test this hypothesis, we determined the ability of mutant p63 proteins to interfere with IKK induced by exogenously expressed wt Np63 in TE1 cells. As shown in Figure 5a, Np63-AEC, Np63-EEC, and Np63-LMS proteins impaired the ability of wt Np63 to induce IKK mRNA, indicating their capacity to act in a dominant-negative manner. To further confirm this observation, we analyzed the expression levels of IKK protein in TE1 cells transfected with Np63 in the presence of mutant Np63 proteins (Figure 5b). Densitometric quantification of IKK expression levels confirmed that Np63-FS (EEC) and Np63-536 (AEC) exert a dominant-negative activity, that Np63-306 (EEC) has low interference potency, that and Np63-639 (SHFM) does not interfere at all with Np63-induced IKK expression. The dominant-negative activity of p63 mutants is also demonstrated by their ability to block differentiation-induced activation of IKK reporter transcription (Figure S3).

To confirm that Np63-AEC proteins function as dominant-negative molecules, we analyzed the epidermis of an AEC patient carrying the I537T mutation. We determined the expression of IKK, K1, and Ki-67 by immunohistochemistry on the lesional skin. As shown in Figure 5c, IKK expression is drastically reduced in AEC skin as compared with normal skin of a control subject (Figure 5c, left panels). Furthermore, we observed delayed K1 expression, which is present only in the upper layers of AEC skin, and an increase in the expression of the proliferation marker Ki-67 (Figure 5c, middle and right panels). IKK downregulation in AEC skin is most likely due to reduced transcription, as determined by the analysis of IKK mRNA levels extracted from AEC skin tissue (Figure 5c right histogram).

Taken together, our data demonstrate that Np63 directly induces expression of IKK and that reduced IKK expression, caused by expression of mutant p63 proteins, may contribute to the development of the clinical phenotype observed in ectodermal dysplasias.

Discussion

Epidermal morphogenesis is a tightly regulated process that involves the temporally coordinated activation of specific sets of genes required for keratinocytes to achieve the subsequent differentiation step (Koster and Roop, 2004). In addition, many genes required for the execution of the epidermal stratification program also mediate epithelial–mesenchymal interactions, thus contributing to the formation of epithelial appendages. Analysis of the phenotypes of p63 and IKK knockout mice indicated that p63 and IKK belong to this category of genes (Li et al., 1999; Mills et al., 1999; Takeda et al., 1999; Yang et al., 1999; Koster and Roop, 2004; Sil et al., 2004).

Our results demonstrate that p63 and IKK are part of the same genetic pathway, with p63 directly inducing transcription of IKK. The transcriptional activation of IKK occurs early upon differentiation of cultured keratinocytes. The subsequent translocation of IKK to the nucleus is required for the role of IKK in keratinocyte differentiation. All three Np63 isoforms and, to a lesser extent, TAp63 can transcriptionally activate IKK when overexpressed. However, experiments performed in primary human keratinocytes, in which Np63 was selectively downregulated, clearly indicate that Np63 is required for the induction of IKK during keratinocyte differentiation, confirming the essential role of Np63 in the regulation of proliferation and differentiation of mature keratinocytes (Truong et al., 2006; Koster et al., 2007). The role of IKK as a critical regulator of keratinocyte proliferation was further demonstrated by its ability to rescue normal cell cycle exit in Np63-downregulated cells. The same pathway was independently identified by Candi et al. (2006); however, their data suggest that both classes of p63 isoforms, TA and N, could induce IKK expression when overexpressed in SAOS2 cells. This apparent discrepancy with our data can be explained by the use of different cell types.

The EEC, SHFM, AEC, and LMS syndromes are caused by mutations in the p63 gene. These syndromes show clinical variability, with sparse hair, dry skin, pilosebaceous gland dysplasia, and oligodontia as well as variable abnormalities of hands and feet, ranging from severe ectrodactyly in EEC patients to mild defects in AEC patients (Brunner et al., 2002; Rinne et al., 2006). We hypothesized that alterations in IKK expression levels, caused by the inability of mutant p63 proteins to activate IKK transcription at critical steps during epidermal development, may contribute to the development of some of the clinical phenotypes observed in ectodermal dysplasia patients. Indeed, our data demonstrate that most of the mutant p63 proteins used in this study displayed an altered ability to induce transcription from an IKK reporter and to induce expression of IKK mRNA. These defects are particularly evident in Np63 proteins carrying mutations found in patients with AEC, which display skin abnormalities, and less in Np63 proteins carrying mutations found in ectodermal dysplasias lacking a skin phenotype (SHFM). Moreover, a subset of mutant Np63 proteins expressed in EEC (Np63FS), AEC (Np63-536), or LMS (Np63DAA) patients exhibit a dominant-negative activity toward the induction of IKK transcripts by wt Np63. This effect correlates with the observation of the heterozygous nature of p63-dependent ectodermal dysplasias supporting the hypothesis that some clinical defects observed in ectodermal dysplasia patients may be due to the ability of mutant p63 proteins to prevent gene activation by wt p63. Recent observations confirm that the selective and conditional downregulation of Np63 in the epidermis of newborn mice results in IKK mRNA downregulation and in a clinical phenotype similar to that observed in AEC syndrome (Koster et al., 2007). Furthermore, IKK downregulation was also observed in the lesional skin of AEC patients at both protein and mRNA levels (Figure 5c), further reinforcing the idea that IKK activation by p63 may play a role in the pathogenesis of skin defects in ectodermal dysplasias. Although additional in vivo models (for example, AEC knockin mice) are necessary to confirm our conclusions, the data reported in this study suggest that IKK may act as a critical p63 downstream target gene in the regulation of epidermal morphogenesis and in the development of a subset of ectodermal dysplasias.

Materials and Methods

Cell lines

U2OS (wt-p53 osteosarcoma cells) were grown in DMEM containing 10% regular fetal bovine serum and antibiotics (100 U ml^-1 penicillin and 10 mg ml^-1 streptomycin) at 37 °C. TE1 cells (esophageal SCC cell line) were cultured in RPMI medium containing 10% regular fetal bovine serum and antibiotics (100 U ml^-1 penicillin and 10 mg ml^-1 streptomycin) at 37 °C. Primary mouse keratinocytes were isolated from newborn mice and cultured at 37 °C in low calcium (0.05 mM CaCl₂) keratinocyte basal medium (Clonetics, San Diego, CA) and EGF (10 ng ml^-1). Cryopreserved NHEKs were obtained from Clonetics and grown on calf skin collagen (Sigma, Milan, Italy) coated dishes in serum-free keratinocyte basal medium. Third-passage NHEK cells were used for transfection experiments.

Reporter plasmids, transient transfections, viral infection, and luciferase assays

Expression plasmids encoding for TAp63, TAp63, TAp63, Np63, Np63, Np63, and Np63 mutants L518V and Q536L (AEC), FS (1689insA) and C306R (EEC), 1743 DelAA (LMS), and E639X (SHFM) were obtained from L. Guerrini (University of Milan, Italy). The p53-encoding plasmid was a kind gift of M. Oren (The Weizmann Institute). IKK-expressing adenovirus was already described (Hu et al., 1999). Viral infections were conducted by incubating primary keratinocytes in serum- and Ca²⁺-free Eagle's minimum essential medium with the appropriate adenovirus at a multiplicity of infection of 50 for 3 to 4 hours, followed by culture in normal growth medium. Under these conditions, 90% of primary mouse keratinocytes were infected, as confirmed by X-gal staining in cells infected with Ad-LacZ.

To generate the IKK-luc reporter plasmid, normal human genomic DNA was amplified with primers (hIKKprom sense 5'-CAGTGCTCAACATTCTGGTTGC-3' and hIKKprom antisense 5'-GCCTCAGGTTCCACAGTTGT-3') spanning the -1,182 to +45 region relative to the IKK transcription initiation site. The PCR product was cloned into the SmaI site of the pGL3 basic plasmid (Promega Inc., Madison, WI) and verified by sequencing. The analysis of putative p53 responsive elements was performed using PatSearch software (http://bighost.area.ba.cnr.it/BIG/PatSearch/) and the following strings:

p1=rrrcwwgyyy[3,0,0] 0.13 p2=rrrcwwgyyy[3,0,0];

p1/p2:(p3=rrrcwwgyyyrrrcwwgyyy[3,0,0]

Transfection and luciferase activity assay

For reporter gene assays, cells were transiently co-transfected with the IKK-luc reporter and mammalian expression plasmids encoding for TAp63, TAp63, TAp63, Np63, Np63, Np63, p53, and Np63 mutants L518V, Q536L, FS, C306R, 1743 DelAA, and E639X. Cells were seeded in 12-well plates and transfected with 0.1 g IKK-luc, 0.02 g of pRL-null plasmid, and 1.5 g of the indicated expression plasmids using Lipofectamine 2000 (Invitrogen Inc., Carlsbad, CA). At 24 hours post-transfection, cell extracts were prepared with 1 lysis buffer, and the luciferase activity was measured using the Dual-Luciferase Kit (Promega Inc.) on a Triathler luminometer (Hidex). Results from experiments, performed three times in triplicate wells, are expressed as relative luciferase activity after normalization with the pRL-null plasmid as internal control. Basal activity of the reporter was set to 1. Each histogram bar represents the mean of three independent transfection experiments performed in triplicate. Standard deviations are indicated.

RNA interference

siRNA duplexes targeting Np63, TAp63, and GFP were obtained from MWG-Biotech (Ebersberg, Germany). Sequences are available in the Supplementary Online Material

Primary keratinocytes plated on collagen-coated 35 mm dishes were transfected with 0.5 g of siRNA per dish using Lipofectamine 2000 (Invitrogen).

Antibodies, immunoblotting, and cell fractionation

The Abs used were polyclonal anti-actin (C-11), monoclonal -tubulin (3F3-G2), monoclonal -tubulin (14C11), monoclonal anti-p63 (4A4), polyclonal anti-p63 (H-137), monoclonal anti-p53 (DO-1), and monoclonal anti-HA (F-7), and they were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA); monoclonal anti-IKK and anti-Ki67 Ab was from BD Pharmingen (San Diego, CA); anti-K1 was purchased from Covance (Princeton, NJ).

For immunoblotting assays, cells were lysed in 50 mM Tris, pH 8, 120 mM NaCl, and 0.5%. NP-40, and protein concentration was determined by the Bio-Rad dye-binding assay (Irvine, CA). Total cell extract of 50 g was loaded on denaturing SDS-PAGE gels and blotted onto a polyvinylidene difluoride membrane. Western blot analysis was performed using the enhanced chemiluminescence system (Amersham Pharmacia Biotech Inc., Uppsala, Sweden).

For cyto/nuclear extraction, cells were washed twice in ice-cold PBS. Cells were then pelleted by centrifugation at 1,400 r.p.m. for 5 minutes, washed once in (1 ml) buffer A (10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (pH 7.9), 10 mM KCl, 0,1 mM EDTA, 0,1 mM EGTA (ethylene glycol bis(-aminoethylether)-N,N,N',N',-tetraacetic acid), 1 mM PMSF (phenylmethylsulfonyl fluoride), and 1 mM dithiothreitol), and centrifuged at 10,000 r.p.m. for 10 minutes. Cell pellets were resuspended in buffer A containing 0.1% (v/v) NP-40 for 10 minutes on ice and lysed. Subsequently, cells were centrifuged at 10,000 r.p.m. for 10 minutes. The supernatant was harvested and the nuclear pellet was extracted with buffer C (20 mM Hepes (pH 7.9), 400 mM NaCl, 1 mM EGTA, 1 mM EDTA, 20% (w/v) glycerol, and 1 mM PMSF) for 15 minutes on ice. After incubation, the nuclei were centrifuged at 10,000 r.p.m. for 10 minutes and the supernatant was diluted with four volumes of buffer D (10 mM Hepes (pH 7.9), 50 mM KCl, 0.2 mM EDTA, 25% (w/v) glycerol, 0.5 mM PMSF, and 1 mM DTT).

mRNA expression analysis

For quantitative real-time PCR, total RNA was extracted from cells using the Trizol Reagent (Invitrogen). One microgram of total RNA was reverse transcribed with GeneAmp RNA PCR (Applied Biosystems Italy, Milan, Italy). For RT-qPCR in mouse keratinocytes, the TaqMan Universal PCR Master Mix (Applied Biosystems Italy) was used for PCR amplification of the cDNA using the Applied Biosystem 7300 real-time PCR system. Each mRNA was normalized to the levels of cyclophilin mRNA for each reaction, and the relative quantification of each gene was determined using the comparative C_T method. Primer sequences for IKK and K10 are available in Supplementary Online Material.

For RT-qPCR in human cells, the reverse transcriptase reaction products were used for quantitative real-time PCR amplification, which was performed with the MyiQ Single-Color Real-Time Detection System for quantification with SYBR Green and melting curve analysis (Bio-Rad, Richmond, CA). The hARP gene (human acidic ribosomal protein P0) was used for normalization. Total RNA was extracted from formalin-fixed, paraffin-embedded sections by the Optimum FFPE RNA Isolation Kit (Ambion, Austin, TX) according to the manufacturer's protocol. Primer sequences for IKK, K1, p63, and ARP are available in Supplementary Online Material.

ChIP assay

For ChIP assays, primary human keratinocytes (NHEK) and U2OS cells were used. ChIP assays were performed as described previously (Costanzo et al., 2002). Briefly, after fixing in 1% formaldehyde, cells were lysed for 5 minutes in 50 mM Tris, pH 8.0, 2 mM EDTA, 0.1% NP-40, and 10% glycerol supplemented with protease inhibitors. Nuclei were re-suspended in 50 mM Tris, pH 8.0, 1% SDS, and 5 mM EDTA. Chromatin was sheared by sonication, centrifuged, and diluted 10-fold in 50 mM Tris, pH 8.0, 0.5% NP-40, 0.2 M NaCl, and 0.5 mM EDTA. After pre-clearing with a 50% suspension of salmon sperm-saturated protein A, lysates were incubated at 4 °C overnight with anti-p63, anti-HA, or anti-Myc Abs. Immune complexes were collected with sperm-saturated protein A, washed three times with high salt buffer (20 mM Tris, pH 8.0, 0.1% SDS, 1% NP-40, 2 mM EDTA, and 500 mM NaCl), and three times with 1 Tris/EDTA (TE). Immune complexes were extracted in 1 TE containing 1% SDS, and protein–DNA cross-links were reverted by heating at 65 °C overnight. DNA was extracted by phenol–chloroform, and 1/20th of the immunoprecipitated DNA was used in each PCR reaction.

PCR reactions were performed for 25–35 cycles of denaturation at 95 °C for 45 seconds, annealing at 55–57 °C for 45 seconds, and extension at 72 °C for 45 seconds.

Primer sequences are reported in online Supplementary Material.

Immunohistochemistry and immunofluorescence

Punch biopsy specimens (4 mm) were taken from normal skin of healthy volunteers (n=4). The Declaration of Helsinki protocols were followed and patients gave written approved consent before biopsy. A patient affected by AEC (I537T mutation in p63) was subjected to skin biopsy on the lesional skin after obtaining informed consent from his parents. Paraffin-embedded 5 mm skin sections were kept at 60 °C for 1 hour and then incubated at 80 °C overnight in citrate buffer pH 6 (DAKO Italia, Milan, Italy). The sections were incubated for 1 hour at room temperature with the appropriate dilution of anti-human IKK monoclonal Ab, anti-K1, anti-Ki67, or control mouse Igs. Sections were stained with an avidin–biotin–peroxidase technique (DAKO Italia) by using 3,3'-diaminobenzidine as a substrate (DAKO Italia). For BrdU incorporation assay, we made use of BrdU Immunohistochemistry Assay Kit (Invitrogen) according to the manufacturer's protocol. For indirect immunofluorescence labeling, double labeling was performed on skin biopsies after deparaffination and rehydration. The primary Abs used were monoclonal anti-p63 (4A4) (1:100) and rabbit polyclonal anti-IKK (1:50). Secondary Abs were Alexa 488-conjugated goat anti-rabbit (Molecular Probes, Milan, Italy) and Texas Red-conjugated goat anti-mouse (Molecular Probes). To determine nonspecific binding, staining control experiments with secondary Ab without primary Ab were also performed. Samples were examined with the use of a Zeiss LSM 410 laser-scanning confocal microscope. The picture shown in Figure 2b is representative of four different biopsies taken from four healthy donors.

Conflict of Interest

The authors state no conflict of interest.

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Acknowledgments

We thank Dr Luisa Guerrini for critically reading the manuscript. This work was supported by funds from AIRC, MIUR (PRIN 2005), and the European Commission (FP6 "Active p53" Consortium) to A.C., and from AIRC to S.A. B.M. is supported by an FIRC fellowship. M.K. and D.R.R. were supported by NIH grants and a research grant from the National Foundation for Ectodermal Dysplasias.

SUPPLEMENTARY MATERIAL

Figure S1. Total cell lysate (20 g) from experiment described in Figure 1B right panel was immunoblotted with specific antibodies to detect expression levels of p63 isoforms, p73 isoforms and p53.

Figure S2. Primary mouse keratinocytes were transfected with indicated siRNA and then kept under proliferating or differentiating (2 mM CaCl₂) conditions.

Figure S3. Dominant-negative activity of p63 mutants on differentiation-induced IKK transcription.