The carboxy-terminus of p63 links cell cycle control and the proliferative potential of epidermal progenitor cells

Abstract

The transcription factor p63 (Trp63) plays a key role in homeostasis and regeneration of the skin. The p63 gene is transcribed from dual promoters, generating TAp63 isoforms with growth suppressive functions and dominant-negative ΔNp63 isoforms with opposing properties. p63 also encodes multiple carboxy (C)-terminal variants. Although mutations of C-terminal variants have been linked to the pathogenesis of p63-associated ectodermal disorders, the physiological role of the p63 C-terminus is poorly understood. We report here that deletion of the p63 C-terminus in mice leads to ectodermal malformation and hypoplasia, accompanied by a reduced proliferative capacity of epidermal progenitor cells. Notably, unlike the p63-null condition, we find that p63 C-terminus deficiency promotes expression of the cyclin-dependent kinase inhibitor p21(Waf1/Cip1) (Cdkn1a), a factor associated with reduced proliferative capacity of both hematopoietic and neuronal stem cells. These data suggest that the p63 C-terminus plays a key role in the cell cycle progression required to maintain the proliferative potential of stem cells of many different lineages. Mechanistically, we show that loss of Cα, the predominant C-terminal p63 variant in epithelia, promotes the transcriptional activity of TAp63 and also impairs the dominant-negative activity of ΔNp63, thereby controlling p21(Waf1/Cip1) expression. We propose that the p63 C-terminus links cell cycle control and the proliferative potential of epidermal progenitor cells via mechanisms that equilibrate TAp63 and ΔNp63 isoform function.

Keywords: Cell cycle; Epithelia; Mouse; Proliferation; Stem cells; p21; p63.

Fig. 1.

Alterative splicing at the p63 C-terminus in p63C^−/− mice. (A) Structure and splicing of the p63 C-terminus in WT and ΔC p63 alleles. Arrowheads indicate stop codons in each isoform. The p63^SAM and p63^TI domains are illustrated. Exons are numbered. (B) Expression of p63 C-terminus variants in WT and p63C^−/− epidermis as assessed by PCR. p63C^−/− epidermal cells predominantly express Cα′, which lacks both the p63^SAM and p63^TI domains, whereas WT cells express full-length Cα. (C) Sequencing of Cα′ and Cβ′ expressed in p63C^−/− mice. Stop codons are underlined. Arrows in A indicate the primer pair used to amplify the Cα′ and Cβ′ splicing variants. (D) Structure of p63 isoforms expressed in WT and p63C^−/− mice. p63Cα′ and p63Cβ′ isoforms are collectively referred to as p63ΔC. DBD, DNA-binding domain; OD, oligomerization domain. (E) qPCR analysis of the N-terminus of the p63 gene in WT and p63C^−/− epidermal cells. Shown is expression of TAp63 relative to ΔNp63, with the ΔNp63 level in WT being set to 1.0. Error bars indicate s.d. (F) Western blot with anti-p63 antibody of E14.5 whole epidermal cell extracts from WT, p63C^−/− and p63C^+/− mice. Asterisk indicates non-specific signal. Full, full-length ΔNp63α; ΔC, ΔNp63ΔC.

Fig. 2.

Craniofacial and skeletal abnormalities in p63C^−/− mice. (A) Gross appearance of the head of E17.5 WT, p63C^−/− and p63-null mice. Whereas p63-null mice show cleft lip, p63C^−/− mice frequently lack this characteristic as indicated by asterisks. (B) H&E staining of coronal sections of E17.5 WT and p63C^−/− mouse heads showing the absence and presence, respectively, of cleft of the secondary palate. nc, nasal cavity; p, palatal shelf; t, tongue. Scale bar: 250 µm. (C) Skeletal staining of E17.5 WT, p63C^−/− and p63-null mice showing relatively well developed forelimbs and hindlimbs in p63C^−/− mice compared with those in p63-null mice. (D) Enlarged views of forelimbs and hindlimbs of WT and p63C^−/− mice. s, scapula; h, humerus; r, radius; u, ulna; d, digits; fe, femur; fi, fibula; t, tibia.

Fig. 3.

Epidermal hypoplasia in p63C^−/− mice. (A) Representative E17.5 WT and p63C^−/− mouse embryos (top) and an enlarged view of the p63C^−/− mouse abdomen showing severe skin erosion (bottom). (B) H&E staining of E17.5 WT and p63C^−/− mouse epidermis. Note that p63C^−/− mouse epidermis is significantly thinner than that of WT and lacks hair follicle development. (C) Changes in epidermal thickness during development (mean±s.d.). *P<0.01; n.s., not significant. (D) Immunofluorescence of WT and p63C^−/− mouse epidermis with the indicated antibodies, counterstained with Hoechst 33342 (DNA, blue). Arrows indicate p63-positive cells expanding into the suprabasal layers in WT epidermis. Lori, loricrin; Inv, involucrin. (E) Immunofluorescence of E15.5 WT and p63C^−/− mouse epidermis with anti-p63 and anti-phosphorylated p63 (pp63) antibodies, counterstained with Hoechst 33342. Arrows indicate high p63-positive cells with low phosphorylation in the basal layer of the p63C^−/− mouse epidermis. (F) Western blot of whole epidermal cell extracts from E15.5 WT and p63C^−/− mice. The membrane was probed first with anti-pp63 antibody, stripped and then reprobed with anti-pan-p63 antibody. Full, full-length ΔNp63α; ΔC, ΔNp63ΔC. Scale bars: 50 µm.

Fig. 4.

Reduced proliferative capacity of p63C^−/− epidermal cells. (A) Immunofluorescence of E15.5 WT and p63C^−/− mouse epidermis with anti-BrdU and anti-p63 antibodies, counterstained with Hoechst 33342. Arrows indicate representative p63⁺ BrdU⁺ epidermal progenitor cells. Dotted lines indicate the epidermal-dermal border. (B) Quantification of A (mean±s.e.m.). *P<0.01. (C) Epidermal cells were isolated from E14.0 WT and p63C^−/− mouse embryos, seeded at clonal density in Matrigel-coated dishes and cultivated for 7 days. Inset shows high expression of involucrin (red) in p63C^−/− epidermal cells, which is absent in the WT counterpart. Nuclei were counterstained with Hoechst 33342. (D) Proliferation of WT and p63C^−/− mouse epidermal cells at day 5 of cultivation. Shown are percentages of CK⁺ Ki67⁺ epidermal cells (mean±s.e.m.). *P<0.01. CK refers to pan-cytokeratin. (E) Staining of WT and p63C^−/− mouse epidermal cells with Rhodamine B at 3 weeks of culture. No macroscopically visible clones were detected in p63C^−/− cell cultures. Scale bars: 25 µm in A,C; 1 cm in E.

Fig. 5.

Upregulation of p21^Waf1/Cip1 expression in p63C^−/− mouse epithelia. (A) Immunofluorescence of E15.5 WT and p63C^−/− mouse epidermis with anti-p21^Waf1/Cip1 and anti-pan-cytokeratin (CK) antibodies, counterstained with Hoechst 33342. Dotted lines indicate epidermal-dermal border. (B) Western blot of whole epidermal cell extracts from E15.5 WT and p63C^−/− mice with the antibodies to p21^Waf1/Cip1, p27^Kip1 and p57^Kip2. Anti-Tubulin α antibody was used as a loading control. (C) Immunohistochemistry of mouse esophagus with anti-p63 (left) and anti-p21^Waf1/Cip1 (right) antibodies. Data shown are representative of WT and p63C^−/− mice at E15.5 and p63-null mice at E17.5. Arrows indicate epidermal cells with high p21^Waf1/Cip1 expression. Scale bars: 25 µm in A; 50 µm in C.

Fig. 6.

Altered p63ΔC functions in control of p21^Waf1/Cip1 expression. (A-C) Stimulation of p21^Waf1/Cip1 expression by TAp63ΔC. H1299 cells were transfected with 1.0 µg plasmid expressing each p63 isoform. Samples were prepared 24 h after transfection. (A) Reporter assays using the minimal p21^Waf1/Cip1 reporter (mean±s.d.). RLU, relative light unit. (B) qPCR analysis of endogenous p21^Waf1/Cip1 gene expression. Values are mean±s.d. with the basal level set to 1.0. *P<0.05. (C) Western blot with anti-p63 and anti-p21^Waf1/Cip1 antibodies. (D-F) Loss of dominant-negative activity of ΔNp63ΔC against TAp63-dependent transactivation of p21^Waf1/Cip1. (D) Reporter assays using the minimal p21^Waf1/Cip1 reporter. H1299 cells were transfected with 0.3 µg TAp63 plasmid alone or together with an increasing amount of ΔNp63 plasmid at ratios of 100:1 or 30:1 and cells were lysed 24 h after transfection. Data shown are mean±s.d. Transactivation by TAp63 alone was set to 1.0. (E) qPCR analysis of endogenous p21^Waf1/Cip1 gene expression. H1299 cells were transfected with 1.0 µg TAp63 plasmid alone or together with ΔNp63 plasmid at a 50:1 ratio and RNA was extracted 24 h after transfection. Values are mean±s.d. with the basal level set to 1.0. *P<0.05. (F) Western blot analysis of p21^Waf1/Cip1 expression. H1299 cells were transfected with 1.0 µg TAp63 plasmid alone or together with ΔNp63 plasmid at a 20:1 ratio and cells were lysed 36 h after transfection. The same cell extracts were used to verify expression of each p63 isoform (top). (C,F) Anti-tubulin α antibody was used as a loading control.