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. Author manuscript; available in PMC: 2016 Nov 1.
Published in final edited form as: Leukemia. 2015 May 19;29(11):2221–2229. doi: 10.1038/leu.2015.128

Disruption of the 5S RNP–Mdm2 interaction significantly improves the erythroid defect in a mouse model for Diamond-Blackfan anemia

P Jaako 1,2, S Debnath 1, K Olsson 1, Y Zhang 3, J Flygare 1, MS Lindström 4, D Bryder 2, S Karlsson 1
PMCID: PMC4844018  NIHMSID: NIHMS778778  PMID: 25987256

Abstract

Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by haploinsufficiency of genes encoding ribosomal proteins (RPs). Perturbed ribosome biogenesis in DBA has been shown to induce a p53-mediated ribosomal stress response. However, the mechanisms of p53 activation and its relevance for the erythroid defect remain elusive. Previous studies have indicated that activation of p53 is caused by the inhibition of mouse double minute 2 (Mdm2), the main negative regulator of p53, by the 5S ribonucleoprotein particle (RNP). Meanwhile, it is not clear whether this mechanism solely mediates the p53-dependent component found in DBA. To approach this question, we crossed our mouse model for RPS19-deficient DBA with Mdm2C305F knock-in mice that have a disrupted 5S RNP–Mdm2 interaction. Upon induction of the Rps19 deficiency, Mdm2C305F reversed the p53 response and improved expansion of hematopoietic progenitors in vitro, and ameliorated the anemia in vivo. Unexpectedly, disruption of the 5S RNP–Mdm2 interaction also led to selective defect in erythropoiesis. Our findings highlight the sensitivity of erythroid progenitor cells to aberrations in p53 homeostasis mediated by the 5S RNP–Mdm2 interaction. Finally, we provide evidence indicating that physiological activation of the 5S RNP-Mdm2-p53 pathway may contribute to functional decline of the hematopoietic system in a cell-autonomous manner over time.

INTRODUCTION

Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia characterized by macrocytic anemia with selective absence of erythroid precursors, physical abnormalities and cancer predisposition.1–3 DBA is most often caused by mutations in ribosomal protein S19 (RPS19; eS19) and other genes encoding RPs.4–11 Studies in zebrafish and mouse DBA model systems in vivo and human hematopoietic cells in vitro have suggested that the anemia is caused by the activation of p53 generated by the RP deficiency.12–15 Mice that have a missense mutation in Rps19 exhibit dark skin, retarded growth and a reduction in the number of erythrocytes, and all of these features are rescued in a p53-null background.12 Similarly, the lethal hematopoietic phenotype of the transgenic Rps19 knockdown mice is reversed upon loss of p53.15 Additionally, Dutt et al. demonstrated that inhibition of p53 with the small molecule pifithrin alpha rescues the erythroid defect in RPS19-deficient human bone marrow (BM) cell cultures.14

The identification of p53 as one of the mediators of the DBA phenotype could have therapeutic implications for the treatment of DBA and related disorders. However, strong concerns have to be raised toward therapeutic strategies based on direct interference with p53, given its prominent role as an orchestrator of genetic programs leading to cell cycle arrest, senescence and apoptosis.16 Therefore, it is important to delineate the components upstream of p53 activation upon RP deficiency, as these might provide novel disease-specific targets for therapeutic intervention. The level of p53 is negatively regulated by mouse double minute 2 (Mdm2; HDM2 in humans), which functions as an ubiquitin ligase that targets p53 to the proteasome in the absence of stress.17 Cellular stress signals, such as DNA damage and oncogenic insults, disrupt the interaction between Mdm2 and p53 resulting in the stabilization and activation of p53.17 A body of in vitro studies indicates that perturbed ribosome biogenesis activates the p53 response through the nuclear accumulation of RPL5 and RPL11 that in turn bind to Mdm2 and inhibit its ubiquitin ligase function toward p53.14,18–20 More recently, RPL5 and RPL11 were shown to regulate p53 as part of the 5S ribonucleoprotein particle (RNP), in which the 5S ribosomal RNA is also critical for p53 regulation.21,22 Finally, the physiological relevance of the 5S RNP–Mdm2 interaction was further confirmed by the generation of Mdm2C305F knock-in mice that harbor a missense mutation in the zinc finger region of Mdm2 preventing its binding to 5S RNP.23,24 Disruption of the 5S RNP–Mdm2 interaction in these mice attenuated the p53 activation upon chemically induced ribosome biogenesis stress, and shortened the latency of c-Myc-driven tumorigenesis.23 However, the physiological relevance of the 5S RNP-Mdm2-p53 pathway for generation of the erythroid defect of DBA is not known. By intercrossing Rps19-deficient mice15 with the Mdm2C305F knock-in mice,23 we show, in the current study, that the 5S RNP–Mdm2 interaction has a dominant role in mediating the p53 activation upon Rps19 deficiency and its disruption partially improves the erythropoiesis of Rps19-deficient mice in vivo. We also report the unexpected finding that the loss of 5S RNP–Mdm2 interaction per se causes mild anemia by triggering a p53 signature in erythroid progenitor cells. Finally, our results indicate that disruption of the 5S RNP–Mdm2 interaction has a positive impact on the reconstitution capacity of serially transplanted hematopoietic stem cells (HSCs), suggesting that the 5S RNP-Mdm2-p53 pathway may contribute to the functional decline of the hematopoietic system upon replicative stress.

MATERIAL AND METHODS

Mice

Generation of transgenic Rps19 knockdown mice and the Mdm2C305F knock-in mice has been described earlier.15,23 Mice were maintained in C57BL/6 background and litter mate controls were used. Rps19 deficiency was induced by administrating doxycycline in the food (Bio-Serv, Flemington, NJ, USA; 200 mg/kg). Mice were maintained at Lund University animal facility and experiments were performed with consent from the Lund University animal ethics committee.

Blood and BM cellularity

Peripheral blood was collected from the tail vein into Microvette tubes (Sarstedt, Nϋmbrecht, Germany) and analyzed using Sysmex XE-5000 and Sysmex KX-21 hematology analyzers. BM cells were isolated by crushing hip bones, femurs and tibiae in phosphate-buffered saline containing 2% fetal calf serum (Gibco, Grand Island, NY, USA), stained with Türk’s solution (Merck, Darmstadt, Germany) and counted in Bürker chambers, or alternatively counted using the Sysmex KX-21 hematology analyzer.

Flow cytometry

FACS analysis of the myeloerythroid compartment in BM was performed as previously described.15,25 Antibodies used are listed in the Supplementary Table 1. To evaluate lineage distribution between myeloid, B and T cells in the peripheral blood, erythrocytes were removed by sedimentation with Dextran (2%, Sigma-Aldrich, St Louis, MO, USA) and ACK lysis. Following the lysis of erythrocytes, cells were stained for surface markers. For cell cycle analyses, c-Kit-enriched BM cells were cultured for 72 h and 0.5 × 106 cells were stained for c-Kit. BD Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Biosciences, Franklin Lakes, NJ, USA; 554714) was utilized for intracellular staining according to manufacture’s instructions and DNA was stained with DAPI (Sigma-Aldrich). Experiments were performed using FACS Aria cell sorter (BD Biosciences) and LSR II flow cytometer (BD Biosciences), and analyzed using FlowJo software (Tree Star, Ashland, OR, USA, v9.7.6).

Cell culture

c-Kit+ cells were enriched using CD117 MicroBeads and MACS separation columns (Miltenyi, Bergisch Gladbach, Germany), and cultured in StemSpan SFEM medium (StemCell Technologies, Grenoble, France) supplemented with penicillin/streptomycin (Gibco), mSCF (100 ng/ml, PeproTech, Rocky Hill, NJ, USA), mIL-3 (10 ng/ml, PeproTech) and hEPO (2 U/ml, Janssen-Cilag, Neuss, Germany) ± doxycycline (1 µg/ml; Sigma-Aldrich).

Expression analyses

Total RNA was isolated from cultured cells after 48 h using the RNAeasy micro kit (Qiagen, Hilden, Germany). Complementary DNA was transcribed using SuperScript III reverse trancriptase (Life Technologies, Paisley, UK). Real-time PCR reactions were performed using pre-designed assays (Life Technologies) with the exception of Rps19 that was quantified using the SYBR GreenER system (5′-GCAGAGGCTCTAAGAGTGTGG-3′; 5′-CCAGGTCTCTCTG TCCCTGA-3′) according to manufacturer’s instructions (Life Technologies, 11761-500).

Immunoblotting

Whole-cell extracts were prepared by harvesting of cells directly into Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) followed by sheering of the chromatin using a needle and boiling of the extracts. Proteins were separated on ready-made SDS-PAGE gels (Life Technologies). Proteins on gels were transferred to nitrocellulose membranes using a Trans-Blot Turbo machine (Bio-Rad). Equal loading of protein was verified by Ponceau S staining and confirmed by immunoblotting for β-actin. Blots were blocked in 5% milk in phosphate-buffered saline for at least 20 min and subsequently incubated with the appropriate primary antibodies overnight at 4 °C on a shaker. After three 5-min washes with phosphate-buffered saline, the blots were incubated with the appropriate secondary horseradish peroxidase-conjugated antibody at room temperature for 2 h followed by detection using the SuperSignal West Pico reagent (Thermo Scientific, Logan, UT, USA). Antibodies used are listed in the Supplementary Table 2. Immunoblots were quantified using the ImageJ software (NIH, Bethesda, MD, USA).

Statistical analyses

Student’s t-test was used to determine statistical significance, and two-tailed P-values are shown. Cell culture and expression analysis-related n represents individual biological repeat experiments. A minimum of three biological replicate experiments was performed to justify the use of chosen statistical test.

RESULTS

Disruption of the 5S RNP–Mdm2 interaction improves the expansion of Rps19-deficient hematopoietic progenitor cells

To study the relevance of the 5S RNP-Mdm2-p53 pathway in DBA we took advantage of our mouse model for RPS19-deficient DBA.15 This model contains an Rps19-targeting short hairpin RNA (shRNA-B) that is expressed by a doxycycline-responsive promoter located downstream of the Collagen A1 gene, allowing for an inducible and dose-dependent Rps19 downregulation (Figure 1a). All experimental animals were bred homozygous for the M2-rtTA at the Rosa26 locus. Administration of doxycycline to the shRNA-B mice results in a severe erythroid phenotype that is reversed in the p53-deficient background.15 Importantly, the hematopoietic phenotype in these mice is specific to Rps19 downregulation as it can be cured by enforced expression of RPS19.15,26 We intercrossed the shRNA-B mice with the Mdm2C305F knock-in mice that have a point mutation in the zinc finger domain of Mdm2, which prevents its binding to 5S RNP (Figure 1b).23,24 As a consequence, these mice present with an attenuated p53 response upon ribosome biogenesis stress, but retain a normal response to DNA damage.23 As a working model, we hypothesized that if the p53 response in Rps19-deficient mice is mediated through the 5S RNP–Mdm2 interaction, the Mdm2C305F background should reverse the p53 activity and subsequently improve the erythroid phenotype of these mice (Supplementary Figure 1). To confirm that the Mdm2C305F background had no effect on the Rps19 knockdown efficiency in our model system, we quantified the expression of Rps19 in cultures initiated with c-Kit-enriched hematopoietic progenitor cells from heterozygous (B/+; one shRNA-B allele) and homozygous (B/B; two shRNA-B alleles) shRNA-B mice. After 48 h of doxycycline treatment, the B/+ and B/B cells showed on average 45% and 75% reduction in Rps19 expression, respectively, and Mdm2C305F had no significant impact on the Rps19 knockdown efficiency (Figure 1c).

Figure 1.

Figure 1

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Mdm2C305F improves the expansion of Rps19-deficient hematopoietic progenitors in vitro. (a) Schematic overview of the transgenic Rps19 knockdown mouse model. This model contains an Rps19-targeting shRNA (shRNA-B) under the control of doxycycline-regulatable control element downstream of the Collagen A1 gene, and constitutively active M2 reverse tetracycline transactivator (M2-rtTA) under the endogenous Rosa26.15 (b) Schematic overview of the Mdm2C305F knock-in mouse model. This model has a missense mutation in the central zinc finger domain of Mdm2 that disrupts its binding to the 5S RNP.23 (c) Quantitative real-time PCR analysis of Rps19 mRNA levels in cultures initiated with c-Kit-enriched BM cells isolated from heterozygous (B/+) and homozygous (B/B) shRNA-B mice. Samples for RNA extraction were collected after 48 h (n=6 for the groups without doxycycline; n=3 for the groups with doxycycline). (d) Expansion of 0.5 × 106 c-Kit-enriched BM cells in cultures (n=6 for the groups without doxycycline; n=4 for the groups with doxycycline). (e) Cell cycle analysis of cultured c-Kit-enriched BM cells. DNA content analysis was performed after 72 h on c-Kit+ cells (n=3 per group). Dean-Jett-Fox model was used to calculate percentages of cells in G0/G1, S and G2/M phases (shown in brown). Data are presented as mean±s.d.

We have previously shown that induction of Rps19 deficiency impairs expansion of hematopoietic progenitor cells.15 Therefore, we first assessed the effect of Mdm2C305F on the expansion of Rps19-deficient hematopoietic progenitor cells in vitro. After 5 days, the uninduced control cultures with wild-type Mdm2 or homozygous Mdm2C305F showed no difference in cell number (5.04 ± 0.91 × 106 and 5.04 ± 0.83 × 106, respectively; Figure 1d). In contrast, the doxycycline-treated B/+ cultures with wild-type Mdm2 showed a significant reduction in cell number (2.99 ± 0.82 × 106), and this was improved in a dose-dependent manner in the heterozygous and homozygous Mdm2C305F background (3.87 ± 0.56 × 106 and 4.68 ± 0.93 × 106, respectively; Figure 1d). The B/B cultures with wild-type Mdm2 failed to expand and had 0.32 ± 0.12 × 106 cells after 4 days of culture, while the heterozygous and homozygous Mdm2C305F background resulted in significant, partial restoration of cell numbers (0.63 ± 0.21 × 106 and 0.99 ± 0.14 × 106, respectively; Figure 1d).

In order to assess whether the reduced expansion of Rps19-deficient hematopoietic progenitors was mainly due to altered proliferation or apoptosis, we performed DNA content analysis of the B/B cultures using flow cytometry. Induction of Rps19 deficiency resulted in a dramatic increase in the frequency of apoptotic cells as indicated by accumulation of these cells in the sub-G1 cell cycle phase that corresponds to cells with hypodiploid DNA content (Figure 1e). Furthermore, and similar to our previous study demonstrating a delay in G1/S transition in Rps19-deficient erythroblasts in vivo,15 following the exclusion of apoptotic cells in the sub-G1 fraction from our analysis, we observed a significant accumulation of Rps19-deficient cells in the G1 cell cycle phase (Figure 1e). Although the heterozygous Mdm2C305F background resulted in a partial rescue of both viability and cell cycle progression, the extent of rescue in the homozygous Mdm2C305F background was close to complete (Figure 1e).

5S RNP–Mdm2 interaction underlies the p53 activation in Rps19-deficient hematopoietic progenitor cells regardless of the level of Rps19 downregulation

Next, we assessed the impact of Rps19 deficiency on p53 response by quantifying the expression of p53 transcriptional target genes (Cdkn1a, Ptp4a3, Zmat3, Bax, Ccng1 and Phlda3)15 in c-Kit-enriched hematopoietic progenitor cells. Comparison of uninduced cultures with wild-type or homozygous Mdm2C305F background revealed no differences in the expression of the p53 target genes with the exception of Zmat3 that was found to be significantly upregulated by Mdm2C305F (Figure 2a). However, the induction of Rps19 deficiency triggered a profound p53 response that was dependent on the level of Rps19 deficiency. For instance, the fold increase in expression of Cdkn1a (p21) in B/+ and B/B cultures was 1.8 and 4, respectively. Mdm2C305F significantly reduced the expression of p53 target genes in a dose-dependent manner regardless of the level of Rps19 downregulation (Figure 2a).

Figure 2.

Figure 2

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5S RNP–Mdm2 interaction underlies the p53 activation in Rps19-deficient hematopoietic progenitor cells. (a) Quantitative real-time PCR analysis of previously identified p53 transcriptional target genes15 in cultures initiated with c-Kit-enriched BM cells. Samples for RNA extraction were collected after 48 h (n=5 for the groups without doxycycline; n=3 for the groups with doxycycline). (b) Representative immunoblots of Mdm2, p53 and Rps19 of cultures initiated with c-Kit-enriched B/B BM cells. Samples were collected after 48 h. (c) Densitometry analysis of p53 and Rps19 protein shown in b (n=3). (d) Representative immunoblots of Rps6 and p-Rps6 of cultures initiated with c-Kit-enriched B/B BM cells. Samples were collected after 48 h. Data are presented as mean ±s.d.

Analysis of the doxycycline-treated B/B cells revealed a robust downregulation of Rps19 protein (Figure 2b and c). Furthermore, correlating with the activation of p53 transcriptional response, doxycycline-treated B/B cells with wild-type Mdm2 showed accumulation of the p53 protein as well as its transcriptional target Mdm2 (Figure 2b and c). Remarkably, a homozygous Mdm2C305F background reversed the accumulation of p53. Finally, in agreement with previous studies,27,28 Rps19-deficient cells showed a decrease in the total level of Rps6, but the pool of phosphorylated Rps6 was increased (Figure 2c). Interestingly, Mdm2C305F had no effect on the phosphorylation of Rps6.

Disruption of the 5S RNP–Mdm2 interaction significantly improves the erythroid defect in Rps19-deficient mice

To study the physiological relevance of our in vitro findings, we transplanted BM from the B/B mice into lethally irradiated wild-type recipients (Figure 3a). We chose this experimental strategy as we have previously shown that doxycycline treatment of the B/B mice results in Rps19 haploinsufficiency and the resulting hematopoietic phenotype is autonomous to the blood system.15 Furthermore, this strategy allowed us to monitor the impact of Mdm2C305F on the hematopoietic recovery from transplantation before the onset of Rps19 deficiency. To assess whether Mdm2C305F had an effect on hematopoiesis per se, we analyzed the peripheral blood 1 month after transplantation. No obvious differences in hematological parameters were observed between the recipients transplanted with control or heterozygous Mdm2C305F BM (Figure 3b, Supplementary Table 3). By contrast, when compared with mice with wild-type Mdm2, the recipients with homozygous Mdm2C305F BM showed a significant decrease in the number of erythrocytes (93% of the recipients with wild-type Mdm2), hemoglobin concentration (95% of the recipients with wild-type Mdm2), number of white blood cells (74% of the recipients with wild-type Mdm2) and macrocytosis (Figure 3b). No differences in the number of reticulocytes were observed (Supplementary Table 3).

Figure 3.

Figure 3

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Mdm2C305F ameliorates the anemia in Rps19-deficient mice. (a) Experimental strategy to assess the effect of Mdm2C305F on Rps19-proficient hematopoietic recovery following transplantation and on Rps19-deficient hematopoiesis. One million uninduced unfractionated BM cells were transplanted in 300 µl PBS into the tail vein of lethally irradiated (900 cGy) wild-type recipients. (b) Erythrocyte number, hemoglobin concentration, mean corpuscular volume (MCV), white blood cell and platelet numbers before the administration of doxycycline (n=25, 24 and 33 for the (B/B; +/+), (B/B; C305F/+) and (B/B; C305F/C305F) groups, respectively). (c) Erythrocyte number, hemoglobin concentration, MCV, reticulocyte, white blood cell and platelet numbers 2 weeks after doxycycline administration (n=28, 20, 19 and 19 for the (+/+; +/+), (B/B; +/+), (B/B; C305F/+) and (B/B; C305F/C305F) groups, respectively). (d) BM cellularity 2 weeks after doxycycline administration (n=27, 18, 18 and 17 for the (+/+; +/+), (B/B; +/+), (B/B; C305F/+) and (B/B; C305F/C305F) groups, respectively). Data are presented as mean ±s.d.

Next the recipient mice were administered doxycycline for 2 weeks in order to induce Rps19 deficiency. The onset of Rps19 deficiency leads to a rapid reduction in blood cellularity that is rescued in the p53-null background.15 As Rps19-deficient mice are partly able to compensate for the erythroid defect caused by the induction of Rps19 deficiency, monitoring the blood and BM cellularity during the first weeks of doxycycline administration provides a valid model to assess the effect of Mdm2C305F on the erythroid recovery of Rps19-deficient mice.25 Doxycycline administration to the recipients transplanted with B/B BM cells resulted in a robust reduction in the number of erythrocytes (58% of control), reticulocytes (67% of control) and hemoglobin concentration (59% of control; Figure 3c, Supplementary Table 4). Heterozygous and homozygous Mdm2C305F background led to a significant increase in all erythroid parameters, demonstrating the physiological relevance of the 5S RNP-Mdm2-p53 pathway for the erythroid defect in Rps19-deficient mice (Figure 3c, Supplementary table 4). In addition to improved erythropoiesis, the recipients with Mdm2C305F showed an increase in the number of platelets and total BM cellularity, further demonstrating the more pronounced hematological recovery after induction of Rps19 deficiency in these mice (Figure 3c and d). Finally, despite the reduction in white blood cells prior to doxycycline administration by Mdm2C305F (Figure 3b), Mdm2C305F had no negative effect on white blood cell number in the recipients with Rps19-deficient BM (Figure 3c).

Mdm2C305F knock-in mice show selective defect in erythropoiesis

Given the negative impact of Mdm2C305F per se on the hematopoietic recovery after transplantation (Figure 3b), we decided to characterize the hematopoietic phenotype of the Mdm2C305F knock-in mice in more detail. Indeed, careful elucidation of the hematopoietic phenotype caused by Mdm2C305F is essential in order to estimate the significance of the 5S RNP-Mdm2-p53 pathway on Rps19-deficient erythropoiesis (Figure 3c). Peripheral blood analysis revealed no significant differences between control and heterozygous Mdm2C305F mice (Figure 4a). However, similar to recipients transplanted with the homozygous Mdm2C305F BM (Figure 3b), the homozygous Mdm2C305F knock-in mice showed significant decrease in the number of erythrocytes that was associated with macrocytosis, and decrease in hemoglobin concentration (Figure 4a). In addition to the erythroid phenotype, we observed an increase in the number of platelets (Figure 4a).

Figure 4.

Figure 4

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Mdm2C305F knock-in mice show selective defect in erythropoiesis. (a) Erythrocyte number, hemoglobin concentration, mean corpuscular volume (MCV), platelet number and white blood cell number in primary Mdm2C305F knock-in mice (n=10, 16 and 9 for the (+/+), (C305F/+) and (C305F/C305F) groups, respectively). (b) Bone marrow cellularity (n=8 per group). (c) Frequencies of erythroid-committed preCFU-E and CFU-E progenitor cells, and erythroblasts in the BM of primary Mdm2C305F knock-in mice (n=8 per group). (d) Quantitative real-time PCR analysis of previously identified p53 transcriptional target genes15 in freshly isolated LSK, preGM/GMP and CFU-E progenitor populations (n=4 per group). Data are presented as mean±s.d. EB=erythroblast.

To gain further insights on the nature of the hematopoietic defect in the homozygous Mdm2C305F knock-in mice, we applied FACS strategies that allow for fractionation of HSCs, myeloerythroid progenitors and erythroid precursors in the BM15,25 (Supplementary Figure 2A and 2B). Correlating with the reduced number of erythrocytes in the peripheral blood, BM of the homozygous Mdm2C305F mice showed a distinct reduction in the frequency of erythroid-committed preCFU-E (64% of control) and CFU-E (57% of control) progenitor cells, and more mature erythroblasts in otherwise normocellular BM (Figure 4b and c, and Supplementary Figure 2C).

Although we observed no major differences in p53 response between uninduced (Rps19-proficient) wild-type and homozygous Mdm2C305F cultures of c-kit-enriched hematopoietic stem and progenitor cells (Figure 2a), we wanted to confirm the impact of Mdm2C305F on the p53 pathway in highly purified progenitor subpopulations. Given the strict correlation between the expression of p53 transcriptional target genes and the level of p53 protein (Figure 2a and c), we used the expression of p53 transcriptional targets as an indicator of p53 activity. Strikingly, quantification of the p53 transcriptional targets in freshly isolated immature (Lineage−Sca-1+c-Kit+; LSK), myeloid-committed (preGM/GMP (granulocyte-macrophage progenitor)) and erythroid-committed (colony-forming unit-erythroid (CFU-E)) hematopoietic progenitor cells revealed a relatively selective and pronounced activation of the p53 signature in the erythroid-committed CFU-E progenitors of the homozygous Mdm2C305F knock-in mice (Figure 4d). Corroborating these data, expression of two additional p53 transcriptional target genes Phlda3 and Bax was increased on average 3.9- and 1.7-fold, respectively, in CFU-Es by Mdm2C305F (Supplementary Figure 3).

Mdm2C305F ameliorates the functional decline of the hematopoietic system upon replicative stress

To study the impact of Mdm2C305F on hematopoiesis upon stress, we transplanted lethally irradiated wild-type recipients with unfractionated control or Mdm2C305F BM cells together with unfractionated congenic wild-type support BM (Figure 5a). In contrast to the primary Mdm2C305F knock-in mice, the recipients of Mdm2C305F BM cells developed a profound decrease in white blood cell reconstitution that appeared especially pronounced for the lymphoid lineages (Figure 5b). These results are in line with the significant decrease in the total number of white blood cells upon transplantation of homozygous Mdm2C305F BM (Figure 3b), and demonstrate that the negative impact of Mdm2C305F on hematopoiesis is not restricted to the erythroid lineage upon transplantation-mediated stress. As would be expected due to the competitive nature of the transplantation experiments, BM analysis of the recipients 4–5 months after transplantation revealed no differences in the total BM cellularity or in the frequency of HSCs and multipotent progenitor cells (Figure 5c). Although the frequency of donor-derived multipotent progenitor cells was modestly decreased in the recipients with Mdm2C305F BM, no difference was observed in the frequencies of donor-derived HSCs (Figure 5d).

Figure 5.

Figure 5

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Mdm2C305F ameliorates the functional decline of the hematopoietic system upon replicative stress. (a) Experimental strategy to assess the impact of Mdm2C305F on transplantation-mediated hematopoietic stress. 0.66 million unfractionated donor BM cells were transplanted together with 0.33 million unfractionated support BM cells in 300 µl PBS into the tail vein of lethally irradiated (900 cGy) wild-type recipients. (b) Myeloid (CD11b+), B cell (CD19+) and T cell (CD3+) donor reconstitution in primary recipients 1 and 4 months after transplantation (n=26, 14 and 26 for the (+/+), (C305F/+) and (C305F/C305F) groups, respectively). (c) Bone marrow cellularity and (d) frequencies of total and donor-derived HSCs (CD45.2 LSK CD150+ CD48 −) and multipotent progenitor (MPP; CD45.2 LSK CD150 − CD48 −) cells in primary recipients 4–5 months after transplantation (n=27, 13 and 26 for the (+/+), (C305F/+) and (C305F/C305F) groups, respectively). (e) Experimental strategy to assess the impact of Mdm2C305F on regenerative properties of HSCs. BM from primary recipients were pooled per genotype and 375 FACS-sorted donor HSCs (CD45.2 LSK CD150+ CD48 −) were transplanted together with 0.4 million fresh unfractionated support BM cells in 300 µl PBS into the tail vein of lethally irradiated (900 cGy) wild-type recipients. (f) Myeloid (CD11b+), B cell (CD19+) and T cell (CD3+) donor reconstitution in secondary recipients 5 months after transplantation (n=11 per group and represents the average of two biological experiments). Data are presented as mean±s.d.

To directly assess the effect of Mdm2C305F on regenerative properties of HSCs, we FACS-sorted highly purified donor HSCs (CD45.2 LSK CD150+CD48−) from the control or homozygous Mdm2C305F primary recipients, and transplanted them into lethally irradiated secondary recipients together with fresh unfractionated wild-type BM (Figure 5e). After 5 months, the recipients transplanted with control HSCs showed a robust myeloid reconstitution, whereas the reconstitution of the lymphoid lineages was low (Figure 5f). Surprisingly, the homozygous Mdm2C305F HSCs gave rise to at least equivalent myeloid and lymphoid reconstitution compared with the control HSCs (Figure 5f). Therefore, when taking into account the significant negative impact mediated by MdmC305F in primary transplantations (Figure 5b), these data strongly indicate that Mdm2C305F may ameliorate the functional decline that normally associates with HSCs upon replicative stress.

DISCUSSION

Studies using animal models and primary human hematopoietic cells have suggested that the increased activation of p53 may be the underlying cause of the anemia in DBA.12–15 Our current study confirms the prevailing hypothesis that the p53 activation upon Rps19 deficiency is dominantly mediated through the 5S RNP–Mdm2 interaction and demonstrates for the first time the physiological relevance of the 5S RNP-Mdm2-p53 pathway for the erythroid defect in DBA. Finally, our results suggest that the 5S RNP-Mdm2-p53 pathway may contribute to the replicative stress-associated functional decline of the hematopoietic system.

Our cell culture studies revealed an activation of the p53 pathway upon induction of Rps19 deficiency in c-Kit-enriched HSC and progenitor cells, and that the degree of the p53 response was strictly dependent on the level of Rps19 downregulation. Mdm2C305F caused a significant reduction in the level and activity of p53 regardless of the extent of Rps19 downregulation, demonstrating a dominant role of the 5S RNP–Mdm2 interaction for p53 activation upon Rps19 deficiency. Reduction of the p53 activity in Rps19-deficient hematopoietic stem and progenitor cells by Mdm2C305F normalized their viability and improved their proliferative capacity, resulting in significantly increased expansion of these cells in vitro. Importantly, reduction in p53 activity in cells with haploinsufficient expression of Rps19, a condition similar to DBA, led to a close to complete reversal of the expansion defect.

We and others have previously demonstrated that the hematopoietic phenotype upon Rps19 deficiency is reversed in the absence of p53.12,13,15 However, the use of p53-deficient mice to study the relevance of p53 for the hematopoietic defect in DBA is problematic due to its additional role in restraining hematopoietic stem and progenitor cell proliferation.29,30 Indeed, increased influx of hematopoietic progenitor cells into the erythroid lineage in p53-deficient mice may mask the effect of p53-independent components that contribute to the erythroid defect in Rps19-deficient mice. Given that the Mdm2C305F mice have wild-type p53, this model addresses more accurately the impact of p53 on Rps19-deficient hematopoiesis in absence of the physiological compensatory mechanisms present in the p53-deficient background. The significant improvement of the Rps19-deficient erythropoiesis by Mdm2C305F clearly demonstrates the physiological and cell-autonomous relevance of the 5S RNP-Mdm2-p53 pathway for the disease phenotype in vivo. Interestingly, extent of the erythroid rescue by Mdm2C305F was only partial, which was initially surprising given the robust improvement of the expansion defect of Rps19-deficient hematopoietic stem and progenitors in vitro. This finding can be explained in part by the subsequent identification of the selective erythroid defect caused by Mdm2C305F. Indeed, the homozygous Mdm2C305F knock-in mice showed a reduction in the frequency of erythroid-committed preCFU-E and CFU-E progenitor cells that was associated with a marked increase in the expression of p53 transcriptional target genes at these cellular stages. This erythroid-pronounced activation of the p53 signature was not detected in our cell culture experiments due to the low frequency of preCFU-E and CFU-E progenitors within the c-Kit-enriched HSC and progenitor cells in the homozygous Mdm2C305F mice. Finally, in contrast to the dose-dependent reversal of the p53 response by Mdm2C305F in vitro, the heterozygous and homozygous Mdm2C305F background resulted in comparable erythroid rescue in the Rps19-deficient recipients, further highlighting the negative impact of the Mdm2C305F per se on erythropoiesis. Together with our earlier study locating the most severe erythroid defect in Rps19-deficient mice at the CFU-E stage,15 these data collectively indicate that both activation as well as disruption of the 5S RNP–Mdm2 interaction result in defective erythropoiesis at the level of CFU-E progenitor cell. Erythroid progenitor cells therefore appear especially sensitive to aberrations in p53 homeostasis mediated by the 5S RNP–Mdm2 interaction, with the 5S RNP-Mdm2-p53 pathway representing a likely contributor to the complex pathogenesis of DBA, although the impact of additional and to date unidentified effects caused by inactivation of the Mdm2 zinc finger cannot be entirely ruled out.

In contrast to steady-state hematopoiesis, transplantation of Mdm2C305F BM revealed a significant reconstitution disadvantage compared with the wild-type cells, and this appeared especially pronounced for the lymphoid lineages. These data demonstrate that the defect caused by Mdm2C305F extends beyond the erythroid lineage upon hematopoietic stress. Despite an initial negative impact of Mdm2C305F on hematopoietic regeneration, serial transplantation of purified wild-type and homozygous Mdm2C305F HSCs resulted in comparable myeloid and lymphoid peripheral blood chimerism, suggesting that Mdm2C305F may provide HSCs with a reconstitution advantage over time. As serial transplantation mimics many aspects of normal aging of the hematopoietic system,31 our data therefore implicate that the 5S RNP-Mdm2-p53 pathway may contribute to this process. Interestingly, aging-associated decrease in lymphopoiesis has been attributed to increased levels of the tumor suppressors p16 and p19Arf over time.32 Given that the activation of p53 by p19Arf is at least partially dependent on the 5S RNP–Mdm2 interaction,22 we speculate that disruption of this pathway by Mdm2C305F in serially transplanted HSCs and their progeny could underlie the observed relative increase in their lymphoid reconstitution capacity.

Taken together, our study demonstrates the dominant role of the 5S RNP–Mdm2 interaction as a mediator of the p53 response upon Rps19 deficiency and provides the first physiological evidence that the 5S RNP-Mdm2-p53 pathway contributes to the anemia in DBA, and may also contribute to normal aging of the hematopoietic system.

Acknowledgments

This work was supported by The Swedish Children’s Cancer Society (PJ, SK), The Crafoord Foundation (PJ), The Gunnar Nilsson Cancer Foundation (DB, PJ), The Swedish Research Council (MSL, SK), The Swedish Cancer Society (SK), Hemato-Linné grant (Swedish Research Council Linnaeus), The Tobias Prize awarded by The Royal Swedish Academy of Sciences financed by The Tobias Foundation (SK) and the EU project grants STEMEXPAND and PERSIST (SK).

Footnotes

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu)

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