The p53 tumor suppressor causes congenital malformations in Rpl24-deficient mice and promotes their survival

Abstract

Hypomorphic mutation in one allele of ribosomal protein l24 gene (Rpl24) is responsible for the Belly Spot and Tail (Bst) mouse, which suffers from defects of the eye, skeleton, and coat pigmentation. It has been hypothesized that these pathological manifestations result exclusively from faulty protein synthesis. We demonstrate here that upregulation of the p53 tumor suppressor during the restricted period of embryonic development significantly contributes to the Bst phenotype. However, in the absence of p53 a large majority of Rpl24(Bst/+) embryos die. We showed that p53 promotes survival of these mice via p21-dependent mechanism. Our results imply that activation of a p53-dependent checkpoint mechanism in response to various ribosomal protein deficiencies might also play a role in the pathogenesis of congenital malformations in humans.

FIG. 1.

Silencing of RPL24 triggers a p53-dependent checkpoint response without affecting pre-rRNA processing. A549 cells were transfected with either scrambled (SCR) siRNA or siRNAs specific for indicated RPS for 48 h unless otherwise stated. (A) Transfected cells were fixed and subjected to immunofluorescence staining with anti-p53 antibody (red) and DAPI (blue). Cells that were treated with actinomycin D (5 nM) for 12 h were used as a positive control for p53 staining. (B) Induction of p21 was verified by immunoblot analysis of lysates of transfected cells. The membrane was reprobed with actin antibody as a loading control. (C) BrdU incorporation into DNA of transfected or actinomycin D-treated cells was measured by flow cytometry. Each value represents the mean ± the SD. The SD was calculated on the basis of three independent experiments. (D) The efficacy of silencing in the above experiments was monitored by Northern blot analysis of total RNA using specific cDNA probes. (E) The levels of the indicated RPS and actin in lysates of transfected cells were determined by immunoblotting. (F) Subcellular fractions were prepared from cells that were treated either with SCR siRNA, RPL24 siRNA, or actinomycin D. Equal amounts of ribosome and nonribosome fractions were immunoblotted with antibodies against RPL11, RPL7a, RPS3a, and actin. (G) Lysates of transfected cells were immunoprecipitated with anti-HDM2 antibodies, followed by immunoblotting with anti-RPL11 antibody. Ten percent of the lysates loaded as input was also blotted with anti-RPL11 antibody. (H) Cells were transfected with either SCR or RPL24-1 siRNAs for 24 h and then pulse-chased with l-[methyl-³H]methionine. The positions of the major newly synthesized pre-rRNA intermediates and mature 28S and 18S rRNA species are indicated on the left (upper panel). The efficacy of RPL24 silencing was monitored by Northern blot analysis of total RNA using RPL24 cDNA probe (middle panel). RNA loading was normalized to the expression of 28S rRNA (lower panel). This experiment is a representative of four. (I) Cytosolic (Cyt.) and nuclear fractions (Nucl.) were prepared from untransfected A549 cells and immunoblotted with indicated antibodies.

FIG. 2.

Rpl24^Bst/+ mutation leads to p53 protein accumulation in gastrulating embryos. (A and B) Immunohistochemistry of sections of representative E6.5 (A) and E7.5 (B) embryos of the indicated genotypes with anti-p53 antibody. (C and E) Percentage of cells that were BrdU labeled in E6.5 (C) and E7.5 (E) embryos of the indicated genotypes. (D) Percentage of cells that were in mitosis in the embryonic region of E6.5 wt, Rpl24^Bst/+, and Rps6^+/− embryos. (F) Apoptosis in section of E7.5 wt, Rpl24^Bst/+, Rps6^+/−, and gamma-irradiated wt embryos. (G) Percentages of apoptotic cells in the embryonic part of E7.5 embryos of the indicated genotypes. wt embryos were gamma irradiated (5 Gy) and analyzed 5 h thereafter in panels B, C, E, and F. The error bars in panels C to E and G denote the SD. In panels C to E and panel G, n = 8 per each genotype. Scale bars: 50 μm (A) and 100 μm (B and F).

FIG. 3.

Accumulation of p53 and p21 proteins in Rpl24^Bst/+ embryos during postgastrulation development. (A) Northern blot analysis of total RNA isolated from E10.5, E11.5, and E18.5 wt (lanes 1) and Rpl24^Bst/+ (lanes 2) embryos using Rpl24 cDNA as a probe. RNA loading was normalized to the expression of β-actin mRNA. (B) The levels of Rpl24 protein in lysates of E10.5 and E11.5 wt (lanes 1) and Rpl24^Bst/+ (lanes 2) embryos were determined by immunoblotting with anti-Rpl24 rabbit polyclonal antibody. Membranes were reprobed with antibodies to Rps6, Rpl7a, and actin. (C) Western blots showing the levels of p53 and p21 proteins in lysates from E10.5, E11.5, and E18.5 wt (lanes 1 and 1′) and Rpl24^Bst/+ (lanes 2 and 2′) embryos. For each genotype, lysates of two littermates were analyzed. Reprobing with antibodies to actin served as a loading control. Lysates of E10.5, E11.5, and E18.5 samples were run on different gels but the membranes were immunoblotted at the same time. Actinomycin D-treated wt mouse embryonic fibroblasts served as a positive control for p53 and p21 antibodies (3). (D) Immunohistochemistry of sagittal sections of the entire E10.5 wt, Rpl24^Bst/+, and gamma-irradiated wt embryos with anti-p53 antibody. (E) Sagittal sections of E10.5 embryos of the indicated genotypes were stained with anti-BrdU antibody. (F) Percentage of cells that were BrdU labeled in the liver (L), mandibula (M), medial nasal process (MNP) and neural tube in the tail (NTT) from E10.5 embryos of the indicated genotypes (n = 8 per each genotype). Error bars denote the SD. (G) Apoptosis in sagittal sections of the entire E10.5 wt, Rpl24^Bst/+, and gamma-irradiated wt embryos. (H) Apoptosis in the liver (L), mandibula (M), roofs of hindbrain (RH), neural tube in the trunk (NTR), and neural tube in the tail (NTT) from embryos of the indicated genotypes (higher magnification of sections from panel G). Apoptotic cells were shown by arrows. wt embryos were gamma-irradiated (5 Gy) and analyzed 5 h thereafter in panels D to H. Scale bars: 500 μm (D, E, and G) and 100 μm (H).

FIG. 4.

Rpl24^Bst/+ mutation does not trigger a p53-dependent checkpoint response in adult mice. (A) Sections of the thymus, spleen, small intestine, kidney, and liver from 6-week-old whole-body gamma-irradiated (5 Gy) wt, nonirradiated wt, and Rpl24^Bst/+ mice were stained with either anti-p53 (left) or anti-activated caspase-3 antibody (right). p53-positive or activated caspase-3-positive cells are indicated by arrows. Scale bar, 100 μm. (B) The protein expression of p21 was determined by immunoblot analysis of lysates of the indicated tissues from whole-body gamma-irradiated wt (lanes 1), nonirradiated wt (lanes 2), and Rpl24^Bst/+ (lanes 3) mice. (C) Northern blot analysis of total RNA was isolated from the indicated tissues from wt (lanes 1) and Rpl24^Bst/+ (lanes 2) mice by using Rpl24 cDNA probe. RNA loading was normalized to the level of β-actin. (D) The levels of the indicated ribosomal proteins in lysates of the indicated tissues from wt (lanes 1) and Rpl24^Bst/+ (lanes 2) mice were determined by immunoblotting. (E) Normal proliferation of Rpl24^Bst/+ T cells. Absolute number of stimulated wt and Rpl24^Bst/+ T cells during 72 h in vitro. Error bars indicate the SD. This experiment is a representative of four. (F) The levels of Rpl24 and p21 proteins in lysates of stimulated and unstimulated wt and Rpl24^Bst/+ T cells. Reprobing with antibody to actin served as a loading control in panels B, D, and F.

FIG. 5.

Frequency of adult or embryonic progeny from p53^−/− males and Rpl24^Bst/+; p53^+/− females. For each genotype, percentages of the total number of recovered animals at weaning (A) or E13.5 (C) are indicated. The ratio of males and females of the indicated genotypes at weaning (B) or E13.5 (D) is shown. The numbers of animals recovered (n) were 192 (A and B) and 149 (C and D). Average litter size, five (A and B) and eight (C and D). At E13.5, 5 of 13 Rpl24^Bst/+; p53^−/− females, 1 of 16 Rpl24^Bst/+; p53^−/− males, and 4 of 14 p53^−/− females were exencephalic (*). Dotted lines indicate the expected percentages of mice of each genotype.

FIG. 6.

p53 inactivation suppresses the phenotype of Rpl24^Bst/+ mice. (A) Morphology of representative (i) wt, (ii) Rpl24^Bst/+, and (iii) Rpl24^Bst/+; p53^−/− embryos at E13.5. (B) Apoptosis in roofs of hindbrains (RH), neural tubes in the trunks (NTR), and neural tubes in the tails (NTT) of E10.5 (i) Rpl24^Bst/+, (ii) Rpl24^Bst/+; p53^−/−, and (iii) Rpl24^Bst/+; p53^+/− embryos. Apoptotic cells are indicated by arrows. (C) The phenotype of adult Rpl24^Bst/+ mice is suppressed by p53 inactivation. Shown are photographs of representative (i) wt, (ii) Rpl24^Bst/+, and (iii) Rpl24^Bst/+; p53^−/− mice (n = 16) and Rpl24^Bst/+; p53^+/− mice (n = 54) at 6 weeks of age. A kink in the tail is indicated by the red arrow. (D) Mean body masses of wt (n = 18), Rpl24^Bst/+ (n = 10), Rpl24^Bst/+; p53^−/− (n = 14), Rpl24^Bst/+; p53^+/− (n = 23), and p53^−/− (n = 17) male mice at 6 weeks of age. We analyzed males because only two female Rpl24^Bst/+; p53^−/− mice were recovered. *, P < 0.05 (Mann-Whitney test). (E) Percentage of wt (n = 32), Rpl24^Bst/+ (n = 30), Rpl24^Bst/+; p53^+/− (n = 32), and p53^+/− (n = 32) mice with the normal pupillary reflex. **, P = 0.006 (chi-square test). Equal numbers of males and females were analyzed. (F) The levels of Rpl24 protein in lysates of E10.5 wt, Rpl24^Bst/+, and Rpl24^Bst/+; p53^−/− embryos were determined by immunoblotting with Rpl24 rabbit polyclonal antibody. Membranes were reprobed with antibodies to Rpl7a, Rps3a, and actin. (G) RNA was isolated from E10.5 embryos of the indicated genotypes. A real-time PCR using 47S rRNA probes was performed in triplicate. The SD was calculated on the basis of three independent experiments. (H) Real time RT-PCR analyses of 84 selected p53 target genes. Eight genes were upregulated more than twofold in E10.5 Rpl24^Bst/+ embryos compared to wt embryos. The SD was calculated on the basis of three independent experiments. (I) Transversal sections of the trunk neural tube of E10.5 wt and Rpl24^Bst/+ embryos were costained with a c-kit and anti-Bax (left panel) or anti-p53 and anti-Bax (right panel) antibodies and analyzed by fluorescence microscopy. Nuclei are stained with DAPI (blue). (J) Transversal sections of the trunk neural tube of E10.5 wt and RPL24^Bst/+ embryos were stained with p53 antibody (red) and B23 (green) and analyzed by confocal laser scanning microscope. Error bars in panels D, G, and H indicate the SD. Scale bars: 1 mm (A), 100 μm (B), 50 μm (I), and 5 μm (J).

FIG. 7.

p21 promotes survival of Rpl24^Bst/+ mice. (A) The phenotype of adult Rpl24^Bst/+ mice is not suppressed by p21 inactivation. Shown are photographs of representative (i) wt, (ii) Rpl24^Bst/+, and (iii) Rpl24^Bst/+; p21^−/− mice (n = 13) and p21^−/− mice (n = 29) at 6 weeks of age. (B) Mean body masses of wt (n = 18), Rpl24^Bst/+ (n = 10), Rpl24^Bst/+; p21^−/− (n = 13), and p21^−/− (n = 29) male mice at 6 weeks of age. (C and D) Frequency of adult progeny from p21^−/− males and Rpl24^Bst/+; p21^+/− females. The number of animals recovered (n) was 204. Average litter size, five. (C) For each genotype the percentages of the total number of recovered animals at weaning are indicated. (D) Ratio of males and females of the indicated genotypes at weaning. Dotted lines indicate the expected percentages of mice of each genotype. (E) Western blot showing the levels of p21 protein in lysates from embryos of the indicated genotypes at E10.5. The membrane was reprobed with antibodies to actin. (F) Percentages of apoptotic cells in the neural tube in the tail (NTT) and roofs of hindbrain (RH) from embryos of the indicated genotypes (n = 8, for each genotype). Error bars in panels B and F indicate the SD. (G) Model explaining our results. Rpl24-deficient ribosomes in the mouse embryos trigger Rpl11-dependent p53 induction. Activated p53 promotes survival of these mice via p21-dependent mechanism and causes congenital malformations independently of p21, most likely by inducing apoptosis.