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Mutant p63 causes defective expansion of ectodermal progenitor cells and impaired FGF signalling in AEC syndrome.

Ferone G, Thomason HA, Antonini D, De Rosa L, Hu B, Gemei M, Zhou H, Ambrosio R, Rice DP, Acampora D, van Bokhoven H, Del Vecchio L, Koster MI, Tadini G, Spencer-Dene B, Dixon M, Dixon J, Missero C - EMBO Mol Med (2012)

Bottom Line: The AEC mutation exerts a selective dominant-negative function on wild-type p63 by affecting progenitor cell expansion during ectodermal development leading to a defective epidermal stem cell compartment.These phenotypes are associated with impairment of fibroblast growth factor (FGF) signalling resulting from reduced expression of Fgfr2 and Fgfr3, direct p63 target genes.Restoring Fgfr2b expression in p63(+/L514F) epithelial cells by treatment with FGF7 reactivates downstream mitogen-activated protein kinase signalling and cell proliferation.

View Article: PubMed Central - PubMed

Affiliation: Fondazione IRCCS SDN, Napoli, Italy.

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Fgfr2 and Fgfr3 are direct p63 target genesReal-time RT-PCR performed on total mRNA isolated from palatal shelves at E13.5, embryonic skin at E14.5, and from neonatal keratinocytes, reveals reduced expression of Fgfr2b in p63+/L514F tissues compared to +/+ controls. Data are normalized for β-actin mRNA levels and are represented as mean ± SD normalized mRNA levels (palate p-value = 0.0015; n = 9, skin p-value = 0.00044; n = 8; keratinocytes p-value = 0.0098; n = 7) and Fgfr3b (palate p-value = 0.0112; n = 8; skin p-value = 0.0092; n = 8; keratinocytes p-value = 0.0002; n = 6).Immunostaining for Fgfr2 (red) reveals its reduced expression in mutant embryonic skin at E15.5 compared to control. Nuclei are stained in blue. Scale bar: 20 µm.Real-time RT-PCR for Fgfr2 pre-mRNA reveals reduced expression of the primary transcript in p63+/L514F keratinocytes compared to +/+ controls. Data are represented as mean ± SD (p-value = 0.023; n = 3). To control for genomic DNA contamination, reverse transcriptase (RT) was not added in the indicated samples.ChIP assay of mouse keratinocyte chromatin using mouse p63 polyclonal antibodies and rabbit IgG as negative control reveals strong p63 binding to Fgfr2 (+1.8 kb from TSS) and Fgfr3 (−10 kb from TSS) regulatory regions. The result is representative of three independent experiments, and error bars represent standard error (SE).ChIP assay of E14.5 skin chromatin reveals a similar p63 binding to Fgfr2 and Fgfr3 regulatory regions. The control regions (ctr) were at −130 bp from TSS and −8.5 kb from TSS in Fgfr2 and Fgfr3, respectively. Data are represented as mean ± SE and are representative of at least three independent experiments.Luciferase assay of the Fgfr2 regulatory region in H1299 reveals that p63 can enhance the wild-type (WT) region activity but not the mutant (MUT) one in cells devoid of p63. See Supporting Information Table S3 for the p63 binding site mutation.Luciferase assay in H1299 reveals that mutant p63L514F is unable to transactivate the Fgfr2 regulatory region and interferes with wild-type p63 affecting its transactivation ability. Data are represented as mean ± SE and are representative of at least three independent experiments.
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fig04: Fgfr2 and Fgfr3 are direct p63 target genesReal-time RT-PCR performed on total mRNA isolated from palatal shelves at E13.5, embryonic skin at E14.5, and from neonatal keratinocytes, reveals reduced expression of Fgfr2b in p63+/L514F tissues compared to +/+ controls. Data are normalized for β-actin mRNA levels and are represented as mean ± SD normalized mRNA levels (palate p-value = 0.0015; n = 9, skin p-value = 0.00044; n = 8; keratinocytes p-value = 0.0098; n = 7) and Fgfr3b (palate p-value = 0.0112; n = 8; skin p-value = 0.0092; n = 8; keratinocytes p-value = 0.0002; n = 6).Immunostaining for Fgfr2 (red) reveals its reduced expression in mutant embryonic skin at E15.5 compared to control. Nuclei are stained in blue. Scale bar: 20 µm.Real-time RT-PCR for Fgfr2 pre-mRNA reveals reduced expression of the primary transcript in p63+/L514F keratinocytes compared to +/+ controls. Data are represented as mean ± SD (p-value = 0.023; n = 3). To control for genomic DNA contamination, reverse transcriptase (RT) was not added in the indicated samples.ChIP assay of mouse keratinocyte chromatin using mouse p63 polyclonal antibodies and rabbit IgG as negative control reveals strong p63 binding to Fgfr2 (+1.8 kb from TSS) and Fgfr3 (−10 kb from TSS) regulatory regions. The result is representative of three independent experiments, and error bars represent standard error (SE).ChIP assay of E14.5 skin chromatin reveals a similar p63 binding to Fgfr2 and Fgfr3 regulatory regions. The control regions (ctr) were at −130 bp from TSS and −8.5 kb from TSS in Fgfr2 and Fgfr3, respectively. Data are represented as mean ± SE and are representative of at least three independent experiments.Luciferase assay of the Fgfr2 regulatory region in H1299 reveals that p63 can enhance the wild-type (WT) region activity but not the mutant (MUT) one in cells devoid of p63. See Supporting Information Table S3 for the p63 binding site mutation.Luciferase assay in H1299 reveals that mutant p63L514F is unable to transactivate the Fgfr2 regulatory region and interferes with wild-type p63 affecting its transactivation ability. Data are represented as mean ± SE and are representative of at least three independent experiments.

Mentions: To provide further support for Fgfr2b or other FGF signalling components being affected in p63+/L514F embryos, we first measured the expression levels of FGF receptors in mutant and wild-type tissues. Fgfr2b mRNA was significantly reduced in p63+/L514F palatal shelves, skin and primary keratinocytes as compared to controls (Fig 4A and B), whereas its expression was unaffected in p63+/− skin (Supporting Information Fig S6B). Among the other FGF receptors, expression of the epidermal-specific isoform Fgfr3b, which plays a role in promoting both epidermal cell proliferation and differentiation (Mandinova et al, 2009), was significantly reduced in p63+/L514F tissue and cells as compared to p63+/− and wild-type controls (Fig 4A). In contrast, Fgfr1 and Fgfr4 were expressed equally in mutant and wild-type embryonic tissues (Supporting Information Table S2).


Mutant p63 causes defective expansion of ectodermal progenitor cells and impaired FGF signalling in AEC syndrome.

Ferone G, Thomason HA, Antonini D, De Rosa L, Hu B, Gemei M, Zhou H, Ambrosio R, Rice DP, Acampora D, van Bokhoven H, Del Vecchio L, Koster MI, Tadini G, Spencer-Dene B, Dixon M, Dixon J, Missero C - EMBO Mol Med (2012)

Fgfr2 and Fgfr3 are direct p63 target genesReal-time RT-PCR performed on total mRNA isolated from palatal shelves at E13.5, embryonic skin at E14.5, and from neonatal keratinocytes, reveals reduced expression of Fgfr2b in p63+/L514F tissues compared to +/+ controls. Data are normalized for β-actin mRNA levels and are represented as mean ± SD normalized mRNA levels (palate p-value = 0.0015; n = 9, skin p-value = 0.00044; n = 8; keratinocytes p-value = 0.0098; n = 7) and Fgfr3b (palate p-value = 0.0112; n = 8; skin p-value = 0.0092; n = 8; keratinocytes p-value = 0.0002; n = 6).Immunostaining for Fgfr2 (red) reveals its reduced expression in mutant embryonic skin at E15.5 compared to control. Nuclei are stained in blue. Scale bar: 20 µm.Real-time RT-PCR for Fgfr2 pre-mRNA reveals reduced expression of the primary transcript in p63+/L514F keratinocytes compared to +/+ controls. Data are represented as mean ± SD (p-value = 0.023; n = 3). To control for genomic DNA contamination, reverse transcriptase (RT) was not added in the indicated samples.ChIP assay of mouse keratinocyte chromatin using mouse p63 polyclonal antibodies and rabbit IgG as negative control reveals strong p63 binding to Fgfr2 (+1.8 kb from TSS) and Fgfr3 (−10 kb from TSS) regulatory regions. The result is representative of three independent experiments, and error bars represent standard error (SE).ChIP assay of E14.5 skin chromatin reveals a similar p63 binding to Fgfr2 and Fgfr3 regulatory regions. The control regions (ctr) were at −130 bp from TSS and −8.5 kb from TSS in Fgfr2 and Fgfr3, respectively. Data are represented as mean ± SE and are representative of at least three independent experiments.Luciferase assay of the Fgfr2 regulatory region in H1299 reveals that p63 can enhance the wild-type (WT) region activity but not the mutant (MUT) one in cells devoid of p63. See Supporting Information Table S3 for the p63 binding site mutation.Luciferase assay in H1299 reveals that mutant p63L514F is unable to transactivate the Fgfr2 regulatory region and interferes with wild-type p63 affecting its transactivation ability. Data are represented as mean ± SE and are representative of at least three independent experiments.
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fig04: Fgfr2 and Fgfr3 are direct p63 target genesReal-time RT-PCR performed on total mRNA isolated from palatal shelves at E13.5, embryonic skin at E14.5, and from neonatal keratinocytes, reveals reduced expression of Fgfr2b in p63+/L514F tissues compared to +/+ controls. Data are normalized for β-actin mRNA levels and are represented as mean ± SD normalized mRNA levels (palate p-value = 0.0015; n = 9, skin p-value = 0.00044; n = 8; keratinocytes p-value = 0.0098; n = 7) and Fgfr3b (palate p-value = 0.0112; n = 8; skin p-value = 0.0092; n = 8; keratinocytes p-value = 0.0002; n = 6).Immunostaining for Fgfr2 (red) reveals its reduced expression in mutant embryonic skin at E15.5 compared to control. Nuclei are stained in blue. Scale bar: 20 µm.Real-time RT-PCR for Fgfr2 pre-mRNA reveals reduced expression of the primary transcript in p63+/L514F keratinocytes compared to +/+ controls. Data are represented as mean ± SD (p-value = 0.023; n = 3). To control for genomic DNA contamination, reverse transcriptase (RT) was not added in the indicated samples.ChIP assay of mouse keratinocyte chromatin using mouse p63 polyclonal antibodies and rabbit IgG as negative control reveals strong p63 binding to Fgfr2 (+1.8 kb from TSS) and Fgfr3 (−10 kb from TSS) regulatory regions. The result is representative of three independent experiments, and error bars represent standard error (SE).ChIP assay of E14.5 skin chromatin reveals a similar p63 binding to Fgfr2 and Fgfr3 regulatory regions. The control regions (ctr) were at −130 bp from TSS and −8.5 kb from TSS in Fgfr2 and Fgfr3, respectively. Data are represented as mean ± SE and are representative of at least three independent experiments.Luciferase assay of the Fgfr2 regulatory region in H1299 reveals that p63 can enhance the wild-type (WT) region activity but not the mutant (MUT) one in cells devoid of p63. See Supporting Information Table S3 for the p63 binding site mutation.Luciferase assay in H1299 reveals that mutant p63L514F is unable to transactivate the Fgfr2 regulatory region and interferes with wild-type p63 affecting its transactivation ability. Data are represented as mean ± SE and are representative of at least three independent experiments.
Mentions: To provide further support for Fgfr2b or other FGF signalling components being affected in p63+/L514F embryos, we first measured the expression levels of FGF receptors in mutant and wild-type tissues. Fgfr2b mRNA was significantly reduced in p63+/L514F palatal shelves, skin and primary keratinocytes as compared to controls (Fig 4A and B), whereas its expression was unaffected in p63+/− skin (Supporting Information Fig S6B). Among the other FGF receptors, expression of the epidermal-specific isoform Fgfr3b, which plays a role in promoting both epidermal cell proliferation and differentiation (Mandinova et al, 2009), was significantly reduced in p63+/L514F tissue and cells as compared to p63+/− and wild-type controls (Fig 4A). In contrast, Fgfr1 and Fgfr4 were expressed equally in mutant and wild-type embryonic tissues (Supporting Information Table S2).

Bottom Line: The AEC mutation exerts a selective dominant-negative function on wild-type p63 by affecting progenitor cell expansion during ectodermal development leading to a defective epidermal stem cell compartment.These phenotypes are associated with impairment of fibroblast growth factor (FGF) signalling resulting from reduced expression of Fgfr2 and Fgfr3, direct p63 target genes.Restoring Fgfr2b expression in p63(+/L514F) epithelial cells by treatment with FGF7 reactivates downstream mitogen-activated protein kinase signalling and cell proliferation.

View Article: PubMed Central - PubMed

Affiliation: Fondazione IRCCS SDN, Napoli, Italy.

Show MeSH
Related in: MedlinePlus