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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

View larger version (35K): [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.

View larger version (37K): [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 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 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.

View larger version (28K): [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.