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Ribosomal Protein Mutations Result in Constitutive p53 Protein Degradation through Impairment of the AKT Pathway.

Antunes AT, Goos YJ, Pereboom TC, Hermkens D, Wlodarski MW, Da Costa L, MacInnes AW - PLoS Genet. (2015)

Bottom Line: In fact, we find that RP-deficient embryos exhibit the same normal p53 transcription, absence of p53 protein, and impaired p53 response to DNA damage as RP haploinsufficient tumor cells.Recently we reported that RP mutations suppress activity of the AKT pathway, and we show here that this suppression results in proteasomal degradation of p53.By re-activating the AKT pathway or by inhibiting GSK-3, a downstream modifier that normally represses AKT signaling, we are able to restore the stabilization of p53.

View Article: PubMed Central - PubMed

Affiliation: Hubrecht Institute, KNAW and University Medical Center Utrecht, Utrecht, the Netherlands.

ABSTRACT
Mutations in ribosomal protein (RP) genes can result in the loss of erythrocyte progenitor cells and cause severe anemia. This is seen in patients with Diamond-Blackfan anemia (DBA), a pure red cell aplasia and bone marrow failure syndrome that is almost exclusively linked to RP gene haploinsufficiency. While the mechanisms underlying the cytopenia phenotype of patients with these mutations are not completely understood, it is believed that stabilization of the p53 tumor suppressor protein may induce apoptosis in the progenitor cells. In stark contrast, tumor cells from zebrafish with RP gene haploinsufficiency are unable to stabilize p53 even when exposed to acute DNA damage despite transcribing wild type p53 normally. In this work we demonstrate that p53 has a limited role in eliciting the anemia phenotype of zebrafish models of DBA. In fact, we find that RP-deficient embryos exhibit the same normal p53 transcription, absence of p53 protein, and impaired p53 response to DNA damage as RP haploinsufficient tumor cells. Recently we reported that RP mutations suppress activity of the AKT pathway, and we show here that this suppression results in proteasomal degradation of p53. By re-activating the AKT pathway or by inhibiting GSK-3, a downstream modifier that normally represses AKT signaling, we are able to restore the stabilization of p53. Our work indicates that the anemia phenotype of zebrafish models of DBA is dependent on factors other than p53, and may hold clinical significance for both DBA and the increasing number of cancers revealing spontaneous mutations in RP genes.

No MeSH data available.


Related in: MedlinePlus

Early cell death of RP mutant cells requires p53.A) Acridine orange staining allowing visualization of dying cells in wild type or rpS7 mutants at 1 dpf that are uninjected, injected with the p53 MO, or injected with the missense MO. Size bar = 0.25mm. B) Quantification of (A). **p<0.01. C) Acridine orange staining of a rpS7 mutant is observed predominantly in the brain region (arrowhead) or distributed over the surface of the tail (white boxes). D) Scoring results from o-dianisidine staining allowing visualization of hemoglobin-expressing cells in clutches of either 111 embryos (+mis MO) or 177 embryos (+p53 MO) from an rpS7 pairing.
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pgen.1005326.g001: Early cell death of RP mutant cells requires p53.A) Acridine orange staining allowing visualization of dying cells in wild type or rpS7 mutants at 1 dpf that are uninjected, injected with the p53 MO, or injected with the missense MO. Size bar = 0.25mm. B) Quantification of (A). **p<0.01. C) Acridine orange staining of a rpS7 mutant is observed predominantly in the brain region (arrowhead) or distributed over the surface of the tail (white boxes). D) Scoring results from o-dianisidine staining allowing visualization of hemoglobin-expressing cells in clutches of either 111 embryos (+mis MO) or 177 embryos (+p53 MO) from an rpS7 pairing.

Mentions: We measured overall levels of cell death in developing embryos carrying the rpS7 mutation with acridine orange (AO), a stain commonly used to detect cells undergoing apoptosis in zebrafish embryos [36]. Fig 1A and 1B show that at 1 dpf, rpS7 mutant embryos reveal a significantly larger number of apoptotic cells compared to wild type controls. Closer visualization of an rpS7 mutant in Fig 1C reveals that these AO-stained cells are found clustered in the brain area and evenly distributed on the entire surface of the tail. The injection of a translation-blocking morpholino that we have previously demonstrated is specific to zebrafish p53 (p53 MO) but not a missense control morpholino (mis MO), completely rescued the number of cells undergoing apoptosis in the rpS7 mutants, reinforcing a role for p53 in cell death as a result of RP loss (Fig 1A and 1B) [37]. The p53 MO also results in the rescue of morphological phenotypes commonly observed in ribosome biogenesis mutants such as the inflation of the hindbrain vesicle and pericardial edemas, a rescue effect that has been previously demonstrated in other zebrafish models of RP loss (S2 Fig) [15,38]. To determine if the p53 MO was sufficient to rescue the defective hematopoiesis of the mutants we used o-dianisidine, which stains hemoglobin-expressing cells evident at 2 dpf. Staining with o-dianisidine revealed that despite the rescue of apoptotic cells by p53 depletion observed at 1 dpf in the rpS7 mutants, the p53 MO was not able to rescue hematopoietic development to any appreciable degree (Fig 1D).


Ribosomal Protein Mutations Result in Constitutive p53 Protein Degradation through Impairment of the AKT Pathway.

Antunes AT, Goos YJ, Pereboom TC, Hermkens D, Wlodarski MW, Da Costa L, MacInnes AW - PLoS Genet. (2015)

Early cell death of RP mutant cells requires p53.A) Acridine orange staining allowing visualization of dying cells in wild type or rpS7 mutants at 1 dpf that are uninjected, injected with the p53 MO, or injected with the missense MO. Size bar = 0.25mm. B) Quantification of (A). **p<0.01. C) Acridine orange staining of a rpS7 mutant is observed predominantly in the brain region (arrowhead) or distributed over the surface of the tail (white boxes). D) Scoring results from o-dianisidine staining allowing visualization of hemoglobin-expressing cells in clutches of either 111 embryos (+mis MO) or 177 embryos (+p53 MO) from an rpS7 pairing.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4488577&req=5

pgen.1005326.g001: Early cell death of RP mutant cells requires p53.A) Acridine orange staining allowing visualization of dying cells in wild type or rpS7 mutants at 1 dpf that are uninjected, injected with the p53 MO, or injected with the missense MO. Size bar = 0.25mm. B) Quantification of (A). **p<0.01. C) Acridine orange staining of a rpS7 mutant is observed predominantly in the brain region (arrowhead) or distributed over the surface of the tail (white boxes). D) Scoring results from o-dianisidine staining allowing visualization of hemoglobin-expressing cells in clutches of either 111 embryos (+mis MO) or 177 embryos (+p53 MO) from an rpS7 pairing.
Mentions: We measured overall levels of cell death in developing embryos carrying the rpS7 mutation with acridine orange (AO), a stain commonly used to detect cells undergoing apoptosis in zebrafish embryos [36]. Fig 1A and 1B show that at 1 dpf, rpS7 mutant embryos reveal a significantly larger number of apoptotic cells compared to wild type controls. Closer visualization of an rpS7 mutant in Fig 1C reveals that these AO-stained cells are found clustered in the brain area and evenly distributed on the entire surface of the tail. The injection of a translation-blocking morpholino that we have previously demonstrated is specific to zebrafish p53 (p53 MO) but not a missense control morpholino (mis MO), completely rescued the number of cells undergoing apoptosis in the rpS7 mutants, reinforcing a role for p53 in cell death as a result of RP loss (Fig 1A and 1B) [37]. The p53 MO also results in the rescue of morphological phenotypes commonly observed in ribosome biogenesis mutants such as the inflation of the hindbrain vesicle and pericardial edemas, a rescue effect that has been previously demonstrated in other zebrafish models of RP loss (S2 Fig) [15,38]. To determine if the p53 MO was sufficient to rescue the defective hematopoiesis of the mutants we used o-dianisidine, which stains hemoglobin-expressing cells evident at 2 dpf. Staining with o-dianisidine revealed that despite the rescue of apoptotic cells by p53 depletion observed at 1 dpf in the rpS7 mutants, the p53 MO was not able to rescue hematopoietic development to any appreciable degree (Fig 1D).

Bottom Line: In fact, we find that RP-deficient embryos exhibit the same normal p53 transcription, absence of p53 protein, and impaired p53 response to DNA damage as RP haploinsufficient tumor cells.Recently we reported that RP mutations suppress activity of the AKT pathway, and we show here that this suppression results in proteasomal degradation of p53.By re-activating the AKT pathway or by inhibiting GSK-3, a downstream modifier that normally represses AKT signaling, we are able to restore the stabilization of p53.

View Article: PubMed Central - PubMed

Affiliation: Hubrecht Institute, KNAW and University Medical Center Utrecht, Utrecht, the Netherlands.

ABSTRACT
Mutations in ribosomal protein (RP) genes can result in the loss of erythrocyte progenitor cells and cause severe anemia. This is seen in patients with Diamond-Blackfan anemia (DBA), a pure red cell aplasia and bone marrow failure syndrome that is almost exclusively linked to RP gene haploinsufficiency. While the mechanisms underlying the cytopenia phenotype of patients with these mutations are not completely understood, it is believed that stabilization of the p53 tumor suppressor protein may induce apoptosis in the progenitor cells. In stark contrast, tumor cells from zebrafish with RP gene haploinsufficiency are unable to stabilize p53 even when exposed to acute DNA damage despite transcribing wild type p53 normally. In this work we demonstrate that p53 has a limited role in eliciting the anemia phenotype of zebrafish models of DBA. In fact, we find that RP-deficient embryos exhibit the same normal p53 transcription, absence of p53 protein, and impaired p53 response to DNA damage as RP haploinsufficient tumor cells. Recently we reported that RP mutations suppress activity of the AKT pathway, and we show here that this suppression results in proteasomal degradation of p53. By re-activating the AKT pathway or by inhibiting GSK-3, a downstream modifier that normally represses AKT signaling, we are able to restore the stabilization of p53. Our work indicates that the anemia phenotype of zebrafish models of DBA is dependent on factors other than p53, and may hold clinical significance for both DBA and the increasing number of cancers revealing spontaneous mutations in RP genes.

No MeSH data available.


Related in: MedlinePlus