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First published online November 8, 2006
doi: 10.1242/10.1242/jcs.03265

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p63 is upstream of IKK in epidermal development

¹ Biochemistry Laboratory, IDI-IRCCS, c/o University of Rome `Tor Vergata', 00133 Rome, Italy
² Fondazione S. Lucia, via Ardeatina, 00179 Rome, Italy
³ Medical Research Council, Toxicology Unit, Leicester University, Leicester, LE1 9HN, UK

^* Authors for correspondence (e-mail: candi{at}uniroma2.it; gm89{at}le.ac.uk)

Accepted 19 September 2006

Summary

The epidermis, the outer layer of the skin composed of keratinocytes, develops following the action of the transcription factor p63. The mouse Trp63 gene contains two promoters, driving the production of distinct proteins, one with an N-terminal trans-activation domain (TAp63) and one without (Np63), although their relative contribution to epidermal development is not clearly established. To identify the relative role of p63 isoforms in relation to IKK, also known to be essential for epithelial development, we performed both molecular and in vivo analyses using genetic complementation in mice. We found that the action of TAp63 is mediated at the molecular level by direct and indirect transactivation of IKK and Ets-1, respectively. We also found that Np63 upregulates IKK indirectly, through GATA-3. Our data are consistent with a role for p63 directly upstream of IKK in epithelial development.

Key words: p63, Epidermis, Cornification, Skin, Differentiation, Development

Introduction

The epidermis is a multi-layered, stratified epithelium continuously regenerated by terminally differentiating keratinocytes (Candi et al., 2005; Fuchs and Watt, 2003; Owens and Watt, 2003), in which a major role is played by p63 (Yang et al., 1998), a member of the p53 family (Melino et al., 2002; Melino et al., 2003; Yang et al., 2002; Yang and McKeon, 2000). Indeed, mutations in the human TP63 gene cause skin and limb defects (Celli et al., 1999), and p63^–/– mice have no epidermis, no limbs and die at birth as a result of dehydration (Mills et al., 1999; Yang et al., 1999).

The expression of p63 proteins originates from two promoters, giving rise to two distinct isoforms, TAp63 and Np63. In addition, both isoforms undergo alternative splicing at the C-terminus producing different TAp63 and Np63 isoforms, respectively named , ß and , with being the longest. Although p63 is involved in epithelial development (Mills et al., 1999; Yang et al., 1999), the relative contribution of these different isoforms to epidermal formation has been established at the molecular level only partially (Candi et al., 2006; Koster et al., 2004).

The formation of the epidermis requires the action of the IB kinase- (IKK). IKK shows both protein-kinase-dependent and -independent effects; the kinase-independent function is required for epidermal keratinocyte differentiation, skeletal and craniofacial morphogenesis, as shown by the IKK^–/– mice (Hu et al., 1999; Li et al., 1999; Sil et al., 2004). Thus, loss of IKK prevents terminal differentiation of keratinocytes by blocking the expression of late differentiation markers, such as loricrin and filaggrin (Hu et al., 1999; Hu et al., 2001; Li et al., 1999), and expression of IKK, under the basal layer promoter cytokeratin 14 (K14) in IKK^–/– mice, produced efficient rescue of the major morphological abnormalities (Sil et al., 2004). IKK is also required for normal whisker and tooth development, where, in the developing oral cavity, the epithelium invaginates inward into the underlying mesenchyme (Ohazama et al., 2004). This appears to be independent of NF-B. IKK knockout mice also show an abnormal tooth cusp morphology very similar to EdaA1, Edar and Edaradd mutants. Changes in Notch1, Notch2, Wnt7b and Shh gene expression in incisor epithelium of IKK-deficient mice suggest that this IKK function is downstream of EdaA1/Edar/Edaradd and is mediated by Notch/Wnt/Shh signaling pathways. Interestingly, p63 is also required for whisker and tooth development (Laurikkala et al., 2006; Rufini et al., 2006). Here, Bmp7, Fgfr2b, Jag1, Notch1 and Edar transcripts are co-expressed with Np63, and are absent in p63^–/– mice (Laurikkala et al., 2006). The involvement of p63 and IKK in the development of the epidermis, whiskers and teeth, raises the question of the reciprocal molecular relationship between p63 and IKK.

Here, we provide evidence that p63 is directly upstream of IKK in epidermal development. Using HA-tagged p63-inducible Tet-on Saos-2 cell lines (see Gressner et al., 2005), we demonstrate that TAp63 induces the expression of IKK, through the p53-like responsive element on its promoter. In addition, TAp63 also drives the expression of Ets-1, another factor which transactivates the IKK promoter. Finally, we identified four GATA-3 binding sites located in the human IKK proximal promoter, and because both TAp63 and Np63 are able to transactivate the GATA-3 promoter, this provides a further indirect mechanism allowing TAp63 and Np63 to modulate IKK expression. Consequently, we observed that the double complemented mice [transgenic TAp63 and Np63 mice under the keratin 5 promoter crossed into p63^–/– mice, thus named p63^–/–;N;TA, generated in our laboratory (Candi et al., 2006)] show higher levels of epidermal IKK expression compared with p63^–/– mice. This provides evidence for p63 being directly upstream of IKK in epidermal development.

Results and Discussion

IKK is a transcriptional target of p63
In order to elucidate the molecular mechanism through which p63 regulates the formation of the epidermis, we investigated its ability to induce IKK. The human IKK promoter contains three specific p53-like responsive elements that could be recognised by p63 proteins (Fig. 1A). This promoter region was cloned in a plasmid upstream of a luciferase reporter gene. We observed that, in Tet-inducible Saos-2 cells (Candi et al., 2006; Gressner et al., 2005), TAp63 significantly increased luciferase activity in a dose-dependent manner (Fig. 1B, lanes 2-3), whereas Np63 did not (Fig. 1B, lanes 4-5). In addition, TAp63ß and TAp63 also significantly increased luciferase activity (Fig. 1C, lanes 3-4) compared with Np63ß and Np63 isoforms (Fig. 1C, lanes 6-7). Transactivation of the IKK promoter by TAp63 was also demonstrated in HEK293 cells (data not shown). Microarray studies, performed on TAp63- and Np63-inducible Saos-2 cells (Candi et al., 2006; Gressner et al., 2005), have shown that TAp63 (3.2 times the control level by microarray activation; 6.3 times the control level by real-time PCR at 24 hours), but not Np63 (0.3-fold over control level by microarray activation; 1.1 times the control level by real-time PCR at 24 hours) also drives expression of Ets-1 (Candi et al., 2006) (data not shown). Expression of Ets-1 alone in both types of Saos-2 cells activates the IKK promoter (Fig. 1B, lanes 1, 6) and the IKK promoter contains an Ets-1 consensus motif (Fig. 1A). Co-expression of TAp63 with Ets-1 results in a greater enhancement of IKK promoter activity compared with that produced by TAp63 alone (Fig. 1B, lanes 7, 3). However, co-expression of Np63 with Ets-1 did not increase the activity of the IKK promoter over that produced by Ets-1 alone (Fig. 1B, compare lanes 8 and 6).

View larger version (35K): [in this window] [in a new window]   Fig. 1. TAp63 isoforms bind and transactivate the IKK promoter. (A) Schematic structure of the IKK promoter showing the different putative p53-like, Ets-1 and GATA-3 responsive elements. (B) Luciferase assay analysis showing the ability of TAp63 to induce IKK expression. The ratios of IKK promoter to transcription factor are: IKK:TAp63/Np63, 1:0.5; IKK:Ets-1, 1:2; IKK:TAp63/Np63:Ets-1, 1:2:2. The luciferase assay shown was performed in Saos-2 cells, and similar results were also obtained in HEK293 cells (data not shown). Results are shown as mean ± s.d. of three independent experiments. (C) Luciferase assay analysis showing the ability of TAp63 and Np63 isoforms to transactivate IKK in Saos-2 cells. Results are similar to those obtained in Saos-2 Tet-on inducible cells in B. The ratio of IKK promoter to TAp63/Np63 isoforms was 1:2. Results are shown as mean ± s.d. of three independent experiments. (D) ChIP showing the binding of p63 protein to the IKK promoter. Lane 1, marker; lane 2, specific antibody (anti-HA); lane 3, non-specific antibody (nsp); lane 4, input+Dox; lane 5, input–Dox, (representative result of two independent experiments is shown). (E) Real-time PCR performed on Dox (Tet-on) inducible Saos-2 clones. TAp63 isoforms already induce a significant increase of IKK RNA upon 24 hours of Dox treatment, whereas Np63 and p53 do not significantly change IKK RNA levels. Results are mean ± s.d. of three independent experiments.

Using the inducible HA-tagged TAp63 and Np63 Saos-2 cell lines (Gressner et al., 2005), we demonstrated that TAp63 induces IKK through direct binding to the previously described p53-like responsive elements in its promoter (Gu et al., 2004) as demonstrated by chromatin immunoprecipitation (ChIP) experiments (Fig. 1D). Immunoprecipitation, using anti-HA antibodies, of protein-DNA complexes containing HA-tagged TAp63 but not Np63, yielded DNA fragments containing IKK sequences (Fig. 1D, compare lanes 2 in lower and upper panels). No IKK sequence was amplified from immunoprecipitates prepared with the control anti-K5 antibody (Fig. 1D, compare lanes 2 and 3). RT-PCR analysis of IKK expression levels in the inducible TAp63 and Np63 Saos-2 cell lines confirms that expression of all TAp63 isoforms upregulates the transcription of IKK mRNA after 24 hours of induction with Dox, whereas Np63 isoforms and p53 do not (Fig. 1E).

View larger version (37K): [in this window] [in a new window]   Fig. 2. TAp63 isoforms induce IKK protein expression. (A) Western blot in Saos-2 Tet-on inducible cell lines (Dox 2 µg/ml) overexpressing TAp63 or Np63 isoforms. All TAp63 isoforms show an induction of IKK at the protein level (from 1.3- to 3.7-fold). The actin western blot is shown in the lower panel as a loading control. IKK expression was normalised to actin by densitometry and results are reported as fold change over zero time (see graphics below actin bands). (B) Np63 isoforms expression do not change (Np63) or produce a minor downregulation (Np63ß and ) of IKK protein after 12 hours of Dox induction. IKK expression was normalised to actin by densitometry and results are reported as fold change over zero time. (C) Western blot in Saos-2 Tet-on inducible cell lines (Dox 2 µg/ml) overexpressing p53. p53 does not induce IKK at either protein or RNA level (see also Fig. 1) after 6 hours and 12 hours of induction. IKK expression was normalised to actin by densitometry and results are reported as fold change over the level at zero time. All results obtained from the densitometry are shown as mean ± s.d. from three independent experiments.

View larger version (29K): [in this window] [in a new window]   Fig. 3. Regulation of IKK during keratinocyte differentiation. (A) Luciferase assay showing the ability of p63 isoforms to transactivate IKK in HaCaT cells. The ratio IKK promoter to TAp63/Np63 isoforms was 1:2. Results are shown as mean ± s.d. of three independent experiments. (B) Quantitative real-time PCR performed on HaCaT cells induced to differentiate in high Ca2+ for 1 and 3 days. Np63 and K14 mRNA decreases indicating that the cells underwent differentiation. Results are shown as mean ± s.d. from three independent experiments. (C) Western blot using anti-IKK and anti-keratin 1-antibody (K1) on HaCaT cells induced to differentiate in high Ca2+ for 1 to 5 days. K1 is shown as control for keratinocyte differentiation; actin is shown as loading control. (D) Quantification of the western blot by densitometry.

View larger version (47K): [in this window] [in a new window]   Fig. 4. IKK regulation by GATA-3, and overexpression in Np63/TAp63 genetically complemented mice. (A) Galactosidase assay showing the ability of TAp63 and Np63 to activate the GATA-3 promoter. The ratio of GATA-3 promoter to transcription factor is 1:3. The in vitro transcription assay shown was performed in Saos-2 cells, and similar results were also obtained in HEK293 cells (data not shown). Results are shown as mean ± s.d. from three independent experiments. (B) ChIP showing the binding of p53 and p63 proteins to the GATA-3 promoter. Lanes 1 and 2, input; lanes 3 and 4, specific antibody (Sp-IP, anti-GATA-3 and anti p21); lanes 5 and 6, non-specific antibody (Not Sp-IP). A representative result of two independent experiments is shown. (C) Luciferase assay showing the ability of GATA-3 to directly induce the IKK promoter. The ratio of IKK promoter to transcription factor is 1:1 and 1:3. In lanes 8 and 9 the ratio was 1:1:1. The in vitro transcription assay shown was performed in HaCaT cells, and similar results were also obtained in HEK293 cells (data not shown). Results are shown as mean ± s.d. from three independent experiments. (D) Western blot of epidermal cell extracts from p63–/–, p63–/–;TA, p63–/–;N, p63–/–;N;TA mice (see 13). Double p63–/–;N;TA complemented mice, as well as p63–/–;TA mice, show higher levels of IKK expression in vivo compared with p63–/– mice. As loading control we detected actin. Lower panel shows the normalisation of the induction of IKK to actin protein level.

Indirect regulation of IKK by p63 via GATA-3
To determine additional potential indirect mechanisms of p63-mediated regulation of IKK, we analysed the human IKK promoter. The IKK proximal promoter contains several binding sites for transcription factors which play important roles during skin development and differentiation (see supplementary material Table S1 and Fig. S1). Beside Ets-1, we decide to investigate the possible role of GATA-3, an important mediator of skin and hair development (Kaufman et al., 2003), in regulating IKK expression, based on the observation that GATA-3 contains a p53-like consensus binding site in its proximal promoter region (–242 to –262 bp). Unlike the IKK promoter, both TAp63 and Np63 significantly increased luciferase activity of the GATA-3 promoter (Fig. 4A, lanes 2, 5). Interestingly, p63ß and p63 isoforms are less efficient in transactivating this promoter. Using the inducible HA-tagged TAp63 and Np63 Saos-2 cell lines, we demonstrated that both p63 isoforms (p53 included as positive control), directly bind to the putative p53-like responsive element in GATA-3 promoter as demonstrated by the ChIP experiments (Fig. 4B). Immunoprecipitation, using anti-HA antibodies, of protein-DNA complexes containing HA-tagged TAp63 and Np63, yielded DNA fragments containing GATA-3 sequences. No GATA-3 sequence was amplified from immunoprecipitates prepared with the control anti-K5 antibody (compare Not Sp-IP with Sp-IP). As a positive control we also performed ChIP experiments using p21 (Fig. 4B).

View larger version (28K): [in this window] [in a new window]   Fig. 5. Proposed mechanism of action of p63 upstream of IKK. TAp63 and Np63 have different, synergistic functions in the formation of the epidermis; TAp63 exerts its effects, at least in part, by acting directly upstream of IKK (which is also indirectly transactivated via Ets-1 and GATA-3), whereas Np63 can only activate IKK indirectly, at least partly via GATA-3. bm, basal membrane; bl, basal layer of keratinocytes; CE, cornified envelope.

IKK mediates the function of p63 in genetically complemented mice
To elucidate the individual role of p63 isoforms in the development of the epidermis, we generated transgenic mice expressing either TAp63 and/or Np63 under the control of a keratinocyte specific promoter (keratin 5); we then crossed these mice into a p63^–/– background, generating single (p63^–/–;TA and p63^–/–;N) and double complemented (p63^–/–;N;TA) mice (Candi et al., 2006). Supplementary material Fig. S2 shows the histology of the epidermis both in the wild type, knockout and genetically complemented mice. The p63^–/–;N as well as the p63^–/–;N;TA complemented mice showed greater, though still not normal, formation of the epidermis (Candi et al., 2006). We therefore took advantage of these mice to evaluate the in vivo relationship between p63 and IKK.

In the single complemented mice, selective reintroduction of both TAp63 and Np63 was associated with increased IKK expression compared with p63^–/– mice, although the increase was greater with TAp63 complementation (tenfold with TAp63 versus 3.5-fold with Np63; Fig. 4D, lanes 2 and 3). As expected, IKK expression was also enhanced in the double complemented mice (Fig. 4D, lane 4).

The function of p63 in epithelial development is mediated by IKK
TP63 is expressed very early in keratinocyte differentiation (Mills et al., 1999; Yang et al., 1999). The Np63 protein is expressed in the basal layer of the human epidermis (Laurikkala et al., 2006; Nylander et al., 2002) and, in both zebra fish and mice, determines the outgrowth of the epidermis (Laurikkala et al., 2006; Lee and Kimelman, 2002). Recently, a distinct contribution of the TAp63 protein has also been demonstrated in mice (Candi et al., 2006). As yet, however, relatively few downstream targets of p63 are known (Candi et al., 2006; Ihrie et al., 2005; Laurikkala et al., 2006) although one of these is fos, a member of the AP1 complex crucial for skin differentiation (Wu et al., 2003) and another is PERP, crucial for epithelial stratification, being localised in desmosomes (Ihrie et al., 2005). Here, we demonstrate that TAp63 and Np63 regulate expression of the gene encoding IKK, which is also crucial for formation of the epidermis. This regulation is both direct, through one of the three p53 consensus motifs in the IKK promoter, and indirect, (1) through TAp63 induction of Ets-1, and (2) through TAp63 and Np63 induction of GATA-3, which subsequently drives IKK expression (Fig. 5). However, as suggested by recent studies (Ihrie et al., 2005; Koster et al., 2004), and by the limited reversion of the p63-null epidermal phenotype by selective TAp63 and Np63 complementation (Candi et al., 2006), other genes must contribute to the proliferative and differentiation potential of the epidermis.

Conclusion

Within the limits of our experimental model, our overall conclusion is that the p63/IKK pathway is essential for epidermal development. These data are important in understanding the molecular mechanisms of development and maintenance of epithelia and thus have important clinical implications (McKeon, 2004).

Materials and Methods

Cell culture
Primary keratinocytes were isolated as described (Yuspa et al., 1989) from the skin of E19.5 transgenic neonates. Saos-2 cells with doxycycline (Dox)-inducible expression of HA-TAp63a, -b, -g and HA-DNp63a, -b, -g were generated as described previously (Gressner et al., 2005).

Mice
The recombination vector contained the K5 promoter (from Manfred Blessing, Joannes Gutemberg University, Mainz, Germany), the PolyA⁺ signal and N-tagged (hemagglutinin antigen, HA) mouse TAp63a or DNp63a (Breuhahn et al., 2000). Transgenic mice on a C57/B6 background were backcrossed with p63^+/– mice (Yang et al., 1999) to generate genetically complemented mice (Candi et al., 2006). Histology was performed according to standard procedures.

Western blots
HaCaT cells were differentiated by addition of Ca²⁺ and collected after 1 and 3 days of treatment. Saos-2-inducible cells were cultured and treated as described previously (Gressner et al., 2005). Cells were lysed as previously reported (Candi et al., 2006) and after extraction, 50 mg protein were separated by SDS-PAGE and transferred onto PVDF membranes; blots were performed using standard procedures. Primary antibody dilutions were: monoclonal anti-p63 (1:200), polyclonal anti-HA (Y-11, Santa Cruz, 1:100), polyclonal anti-IKK (1:200), polyclonal anti-K14 (1:300). Normalisation was achieved with a polyclonal anti-tubulin (H-235, Santa Cruz, 1:1000 dilution) or with a goat anti-actin antibody (C-11, Santa Cruz, 1:1000 dilution). Proteins were detected using the ECL method.

Real-time PCR assay
RNA was extracted from control and Dox-induced SaOs-2 Tet-on HA-tagged TAp63 and –DNp63 clones, using Trizol (Invitrogen, Carlsbad, CA). Real-time PCR was performed as previously described (Candi et al., 2006). Primer sequences are available upon request. Relative quantification of gene expression was performed using ABI 7500 SDS software v 1.31.

ChIP and transcription assay
This was performed according to a previous published protocol (Munarriz et al., 2004). Samples (1.5x10⁶ cells) were incubated with 2 mg monoclonal anti-HA (16B12, Covance) or monoclonal anti-p63 (Ab4, Neomarkers). The negative control was incubated with 2 mg mouse anti-K5 (Santa Cruz). DNA samples were then analysed with 38 cycles of PCR to amplify IKKa promoter sequences (94°C for 30 seconds, 58°C for 40 seconds, 72°C for 40 seconds). For IKKa amplification we used specific forward and reverse primers (5'-GTGGTTCCGTTCAGCCCT-3' and 5'-TGCTCGCGCGTCTTTG-3'). The resulting product was 188 bp and contained the last two putative p53 consensus sequences as well as the Ets-1 binding motif in the IKKa promoter. The following specific oligos were used to determine p53/p63 binding sites of p21 and GATA-3 genes: p21 site F(5'-ATGTATAGGAGCGAAGGTGCA-3'); p21 site R(5'-CCTCCTTTCTGTGCCTGAAACA-3'); GATA-3 site F(5'-GAATTCCCTCCTGCCTGTCC-3'); GATA-3 site R(5'-CTTCACCTCCACCCCCATCC-3'). For luciferase assay in Saos and HaCaT cells, we used the reporter plasmid containing the luciferase gene under the control of IKK promoter and the expression vectors encoding for TAp63, ß, and Np63, ß, (see legend figures for ratios). When needed, the pcDNA empty vector (Invitrogen) was added to reach the total amount of DNA (400 ng) used in each transfection. In all cases, 10 ng of Renilla luciferase vector (pRL-CMV; Promega, Madison, WI) were co-transfected, as a control of transfection efficiency. Luciferase activities of cellular extracts were measured, by using a Dual Luciferase Reporter Assay System (Promega); light emission was measured over 10 seconds using an OPTOCOMP I luminometer. Efficiency of transfection was normalised using Renilla luciferase activity. For ß-galactosidase assay, cells were transiently transfected with a reporter construct containing GATA-3 promoter (–308pLacZ, kindly provided by J. D. Engel, University of Michigan Medical School, MI) and expression vectors encoding p53, p63 isoforms and pcDNA3.1 (see legend figures for ratios). Each transfection experiment was done in triplicate. 48 hours after transfection, cells were washed with PBS and harvested with 100 µl of lysis buffer (5 mM DTT, 250 mM Tris-HCl pH 8.1 containing protease inhibitors). A total of 100 µg of total cell lysate was incubating with 800 µl substrate solution (2 mg/ml ONPG (Pierce) in 0.1 M Na₂HPO₄, 1 M NaH₂PO₄, 1 M KCl, 1 M MgCl₂ with 20% ß-mercaptoethanol) for 40 minutes at 37°C. The reaction was stopped by addition of 1 M Na₂CO₃ and the absorbance was measured at 420 nm.

Acknowledgments

This work was only possible thanks to generous practical and intellectual help of Pierluigi Nicotera. The work was supported by grants from Telethon to E.C., AIRC, Telethon, EU-Grants EPISTEM (LSHB-CT-019067), IMPALED (QLK3-CT-2002-01956), (Active p53), FIRB, MIUR, MinSan, Telethon and Medical Research Council to G.M.

Footnotes

Supplementary material available online at http://jcs.biologists.org/cgi/content/full/119/22/4617/DC1

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