Fig 1.
Lack of p23 results in decreased epidermal thickness, delayed differentiation and increased rates of proliferation and apoptosis in E18.5 skin.
(a) Representative sections stained with H&E showing epidermal skin (delimited by red line) thinning with almost absent stratum corneum in embryonic p23-/- skin sections along with lower numbers of hair follicles. E, epidermis; HF, hair follicle; SC, stratum corneum. (b) Quantitation of epidermal thickness and (c) number of hair follicle per 100 μm basal membrane (BM) in p23-/- and wild-type E18.5 skin sections; significant decrease of epidermal thickness (*** p<0.001) and hair follicle numbers (** p<0.01) in p23-/- skin (n: WT = 4, p23-/- = 3). (d) Immunohistochemical staining for the differentiation markers loricrin, filaggrin and involucrin shows decrease in staining intensity in p23-/- E18.5 skin sections. (e) TUNEL assay indicates significantly higher apoptotic rate in p23-/- E18.5 skin sections compared to wild-type (*** p<0.001; n: WT = 4, p23-/- = 4). (f) BrdU staining of nuclei indicates increased proliferation in p23-/- skin sections (** p<0.01; n: WT = 7, p23-/- = 7). Black bars in panels a and d indicate 50 μm.
Fig 2.
Expression of p23, GR, Fkbp51 and other GR target genes in E18.5 skin.
(a) Immunohistochemical staining for p23, GR, and Fkbp51 in E18.5 skin sections. Note that GR staining was detected almost exclusively in basal keratinocytes, with very few positive cells in the suprabasal layers, and that GR is also present in the dermal compartment. Fkb51 levels were markedly lower in the epidermis of p23-/- E18.5 embryos, while expression of GR was slightly lower in some but not all p23-/- skin sections compared to wild-type. For each protein and genotype, sections of two different embryos are shown. White bars indicate 50 μm. (b) Representative immunoblot (top panel) of extracts from 4 WT and 3 p23-/- embryos shows reduced GR protein levels in E18.5 epidermis in some but not all p23-/- E18.5 epidermal samples compared to wild-type. The quantitation of two immunoblots with a total of 6 WT and 7 p23-/- samples is shown as a bar graph. A trend to a reduction of GR levels is suggested by the statistical analysis (p = 0.0566), whereas Fkbp51 levels are undoubtedly decreased in p23-/- E18.5 epidermis (*** p<0.001). Immunoblotting for p23 was used to confirm the genotype and GAPDH served as loading control. (c) Relative mRNA levels of several other GR target genes in p23-/- vs. wild-type E18.5 epidermis, quantified by qPCR. Results are representative of three independent experiments, each with the 6 WT and 7 p23-/- samples.
Fig 3.
(a) Total number of MPKs that can be obtained from the epidermis of individual p23-/- E18.5 embryos compared to wild-type is significantly lower (* p<0.05; n: WT = 24, p23-/- = 26). (b) Representative micrographs showing differences in cell confluency after seeding MPKs; bars = 50 μm. (c) Increased growth rate of p23-/- MPKs; * p value at 120 h <0.05 (n: WT = 3, p23-/- = 3). (d, e) Increased apoptosis of p23-/- compared to wild-type MPKs without (d; **** p<0.0001; n: WT = 10, p23-/- = 6) and following UV treatment (e; **** p<0.0001; n: WT = 6, p23-/- = 11).
Fig 4.
Differentiation of p23-/- MPKs in vitro is impaired.
(a) Undifferentiated wild-type and p23-/- MPKs from E18.5 embryos were induced to differentiate with 1.2 mM Ca2+ for 24 h and 48 h. No obvious morphological differences were noted by phase contrast microscopy (bars = 50 μm). (b) Immunofluorescence staining for epithelial markers demonstrates impaired differentiation of p23-/- MPKs. Note that only a subset of cells are expected to express K-10 at the relatively early 24 h time point. K-10, keratin-10.
Fig 5.
Dex differentially affects p23-/- versus wild-type MPKs.
(a) Nuclear localization of GR is impaired in p23-/- MPKs. Immunofluorescence micrographs of cells treated with 100 nM Dex for 3 h and stained with an antibody to GR (bars = 50 μm). (b) Opposite effects of 1 μM Dex on the proliferation of wild-type (* p<0.05) and p23-/- (* p<0.05) MPKs 120 h after seeding (n: WT = 3, p23-/- = 3).
Fig 6.
GR-mediated effects on MPK differentiation are not affected by loss of p23.
(a) Phase contrast micrographs of MPKs induced to differentiate with 1 μM Dex for 48 h. (b) Immunofluorescent staining for the differentiation markers keratin-10 (K-10), E-cadherin and involucrin in wild-type and p23-/- MPKs treated with 1 μM Dex for 48 h (bars = 50 μm).