Abstract
MDM2, an E3 ubiquitin ligase, is an important negative regulator of tumor suppressor p53. In turn the Mdm2 gene is a transcriptional target of p53, forming a negative feedback loop that is important in cell cycle control. It has recently become apparent that the ubiquitination of p53 by MDM2 can be inhibited when certain ribosomal proteins, including RPL5 and RPL11, bind to MDM2. This inhibition, and the resulting increase in p53 levels has been proposed to be responsible for the red cell aplasia seen in Diamond-Blackfan anemia (DBA) and in 5q- myelodysplastic syndrome (MDS). DBA and 5q- MDS are associated with inherited (DBA) or acquired (5q- MDS) haploinsufficiency of ribosomal proteins. A mutation in Mdm2 causing a C305F amino acid substitution blocks the binding of ribosomal proteins. Mice harboring this mutation (Mdm2C305F), retain a normal p53 response to DNA damage, but lack the p53 response to perturbations in ribosome biogenesis. While studying the interaction between RP haploinsufficiency and the Mdm2C305F mutation we noticed that Mdm2C305F homozygous mice had altered hematopoiesis. These mice developed a mild macrocytic anemia with reticulocytosis. In the bone marrow (BM), these mice showed a significant decrease in Ter119hi cells compared to wild type (WT) littermates, while no decrease in the number of mature erythroid cells (Ter119hiCD71low) was found in the spleen, which showed compensated bone marrow hematopoiesis. In methylcellulose cultures, BFU-E colonies from the mutant mice were slightly reduced in number and there was a significant reduction in CFU-E colony numbers in mutant mice compared with WT controls (p < 0.01). This erythropoietic defect was abrogated by concomitant p53 deficiency (Trp53ko/ko). Further investigation revealed that in Mdm2C305F animals, there was a decrease in Lin-Sca-1+c-Kit+ (LSK) cells, accompanied by significant decreases in multipotent progenitor (MPP) cells (p < 0.01). Competitive BM repopulation experiments showed that donor BM harboring the Mdm2C305F mutation possessed decreased repopulation capacity compared to WT BM, suggesting a functional stem cell deficit. These results suggest that there is a fine tuned balance in the interaction of ribosomal proteins with the MDM2/p53 axis which is important in normal hematopoiesis.
Highlights
Diamond-Blackfan Anemia (DBA) is a bone marrow failure syndrome characterized by macrocytic anemia and red cell aplasia, sometimes associated with developmental anomalies, cleft palate, thumb abnormalities and cardiovascular problems [1, 2]
We found that Mdm2C305F mice had distinct abnormalities in erythropoiesis that point to the importance of ribosomal proteins (RPs)/MDM2 binding in regulating red cell production
Erythropoietin levels were higher in mutant mice compared to wild type, but this did not reach statistical significance (p = 0.118). erythrocyte adenosine deaminase (eADA) levels, known to be increased in red blood cells from patients with DBA due to ribosomal protein gene mutations, were normal in red blood cells from Mdm2C305F/C305F mutant mice
Summary
Diamond-Blackfan Anemia (DBA) is a bone marrow failure syndrome characterized by macrocytic anemia and red cell aplasia, sometimes associated with developmental anomalies, cleft palate, thumb abnormalities and cardiovascular problems [1, 2]. In acquired 5q- MDS, patients have a defect in erythroid cell differentiation with macrocytic anemia and red cell aplasia [4]. The gene for a ribosomal protein RPS14 is present in the deleted region and appears to be responsible for the development of the red cell defect [5]. Notably RPL5 and RPL11, bind to MDM2 and block its ability to inhibit p53. This mechanism was demonstrated by the inability of ribosome biogenesis to affect p53 in cells homozygous for a mutant MDM2 with a C305F mutation, which fails to bind these ribosomal proteins (RPs) [17]. Cells homozygous for Mdm2C305F have a normal p53 response to DNA damage but not to ribosomal stress
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