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

Journal of Cell Science 119, 4617-4622 (2006)
Published by The Company of Biologists 2006
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p63 is upstream of IKK{alpha} in epidermal development

Eleonora Candi1,*, Alessandro Terrinoni1, Alessandro Rufini1, Anissa Chikh1, Anna Maria Lena1, Yasuhiro Suzuki2, Berna S. Sayan3, Richard A. Knight3 and Gerry Melino1,2,3,*

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


Figure 1
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  		Fig. 1. TAp63 isoforms bind and transactivate the IKK{alpha} promoter. (A) Schematic structure of the IKK{alpha} promoter showing the different putative p53-like, Ets-1 and GATA-3 responsive elements. (B) Luciferase assay analysis showing the ability of TAp63{alpha} to induce IKK{alpha} expression. The ratios of IKK{alpha} promoter to transcription factor are: IKK{alpha}:TAp63{alpha}/{Delta}Np63{alpha}, 1:0.5; IKK{alpha}:Ets-1, 1:2; IKK{alpha}:TAp63{alpha}/{Delta}Np63{alpha}: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 {Delta}Np63 isoforms to transactivate IKK{alpha} in Saos-2 cells. Results are similar to those obtained in Saos-2 Tet-on inducible cells in B. The ratio of IKK{alpha} promoter to TAp63/{Delta}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{alpha} 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{alpha} RNA upon 24 hours of Dox treatment, whereas {Delta}Np63{alpha} and p53 do not significantly change IKK{alpha} RNA levels. Results are mean ± s.d. of three independent experiments.

 

Figure 2
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  		Fig. 2. TAp63 isoforms induce IKK{alpha} protein expression. (A) Western blot in Saos-2 Tet-on inducible cell lines (Dox 2 µg/ml) overexpressing TAp63 or {Delta}Np63 isoforms. All TAp63 isoforms show an induction of IKK{alpha} 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{alpha} expression was normalised to actin by densitometry and results are reported as fold change over zero time (see graphics below actin bands). (B) {Delta}Np63 isoforms expression do not change ({Delta}Np63{alpha}) or produce a minor downregulation ({Delta}Np63ß and {gamma}) of IKK{alpha} protein after 12 hours of Dox induction. IKK{alpha} 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{alpha} at either protein or RNA level (see also Fig. 1) after 6 hours and 12 hours of induction. IKK{alpha} 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.

 

Figure 3
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  		Fig. 3. Regulation of IKK{alpha} during keratinocyte differentiation. (A) Luciferase assay showing the ability of p63 isoforms to transactivate IKK{alpha} in HaCaT cells. The ratio IKK{alpha} promoter to TAp63/{Delta}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. {Delta}Np63{alpha} 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{alpha} 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.

 

Figure 4
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  		Fig. 4. IKK{alpha} regulation by GATA-3, and overexpression in {Delta}Np63/TAp63 genetically complemented mice. (A) Galactosidase assay showing the ability of TAp63{alpha} and {Delta}Np63{alpha} 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{alpha} promoter. The ratio of IKK{alpha} 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–/–;{Delta}N, p63–/–;{Delta}N;TA mice (see 13). Double p63–/–;{Delta}N;TA complemented mice, as well as p63–/–;TA mice, show higher levels of IKK{alpha} expression in vivo compared with p63–/– mice. As loading control we detected actin. Lower panel shows the normalisation of the induction of IKK{alpha} to actin protein level.

 

Figure 5
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  		Fig. 5. Proposed mechanism of action of p63 upstream of IKK{alpha}. TAp63{alpha} and {Delta}Np63{alpha} have different, synergistic functions in the formation of the epidermis; TAp63 exerts its effects, at least in part, by acting directly upstream of IKK{alpha} (which is also indirectly transactivated via Ets-1 and GATA-3), whereas {Delta}Np63 can only activate IKK{alpha} indirectly, at least partly via GATA-3. bm, basal membrane; bl, basal layer of keratinocytes; CE, cornified envelope.

 

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© The Company of Biologists Ltd 2006