Haematopoietic stem cells require a highly regulated protein synthesis rate

Abstract

Many aspects of cellular physiology remain unstudied in somatic stem cells, for example, there are almost no data on protein synthesis in any somatic stem cell. Here we set out to compare protein synthesis in haematopoietic stem cells (HSCs) and restricted haematopoietic progenitors. We found that the amount of protein synthesized per hour in HSCs in vivo was lower than in most other haematopoietic cells, even if we controlled for differences in cell cycle status or forced HSCs to undergo self-renewing divisions. Reduced ribosome function in Rpl24(Bst/+) mice further reduced protein synthesis in HSCs and impaired HSC function. Pten deletion increased protein synthesis in HSCs but also reduced HSC function. Rpl24(Bst/+) cell-autonomously rescued the effects of Pten deletion in HSCs; blocking the increase in protein synthesis, restoring HSC function, and delaying leukaemogenesis. Pten deficiency thus depletes HSCs and promotes leukaemia partly by increasing protein synthesis. Either increased or decreased protein synthesis impairs HSC function.

Figure 1. Quantifying protein synthesis in haematopoietic cells in vivo

a, OP-Puro incorporation in bone marrow cells in vivo one hour after administration. b-d, OP-Puro (b), HPG (c), and AHA (d) incorporation in bone marrow cells in culture was inhibited by cycloheximide (CHX). d, Bone marrow cells from mice treated with OP-Puro in vivo exhibited normal AHA incorporation in culture, indicating OP-Puro did not block protein synthesis. e,f OP-Puro versus HPG (e; n=4 mice from 2 experiments) or AHA (f; n=3 mice from 3 experiments) incorporation by haematopoietic cells in culture. g, OP-Puro incorporation in CD150⁺CD48⁻LSK HSCs and unfractionated bone marrow cells one hour after administration in vivo. h, Protein synthesis in various haematopoietic stem and progenitor cell populations relative to unfractionated bone marrow cells (n=15 mice from 9 experiments). Extended Data Fig. 1j shows the data from Fig. 1h using a log2 scale. Data represent mean±s.d. Statistical significance was assessed using two-tailed Student’s t-tests (e-f) and differences relative to HSCs (h) were assessed using a repeated measures one way ANOVA followed by Dunnett’s test for multiple comparisons (*, p<0.05; **, p<0.01; ***, p<0.001 relative to bone marrow; ###, p<0.001 relative to HSCs).

Figure 2. Lower rate of OP-Puro incorporation by HSCs does not reflect efflux or proteasomal degradation

a,b, OP-Puro fluorescence in haematopoietic cells from Abcg2-deficient and control mice one hour after OP-Puro administration in vivo (n=4 mice from 3 experiments). c, OP-Puro fluorescence in haematopoietic cells one, three, or twenty-four hours after OP-Puro administration (n=5 experiments). d, OP-Puro fluorescence in haematopoietic cells two hours after bortezomib and one hour after OP-Puro administration in vivo (n=5 mice/treatment from 5 experiments). e, Western blots of 30,000 cells from each haematopoietic cell population from OP-Puro-treated or control mice. f, OP-Puro fluorescence in haematopoietic cells one hour after administering 50mg/kg or 100mg/kg OP-Puro (n=5 mice/dose from 5 experiments). All data represent mean±s.d. To assess the statistical significance of treatment effects within the same cells we performed two-tailed Student’s t-tests (*, p<0.05; **, p<0.01; ***, p<0.001). Differences between HSCs and other cell populations were assessed with a repeated measures one way ANOVA followed by Dunnett’s test for multiple comparisons (#, p<0.05; ##, p<0.01; ###, p<0.001).

Figure 3. HSCs synthesize less protein than most haematopoietic progenitors, even when undergoing self-renewing divisions

a, OP-Puro incorporation in vivo in bone marrow cells in G₀/G₁ versus S/G₂/M. b, Protein synthesis in G₀/G₁ and S/G₂/M cells from haematopoietic cell populations in vivo (n=10 mice from 6 experiments). We were unable to assess OP-Puro incorporation in S/G₂/M HSCs and MPPs in these experiments because these cells are extraordinarily rare in normal bone marrow. c, Protein synthesis in haematopoietic cells after Cy/G-CSF treatment (n=10 mice from 6 experiments). d,e, Protein synthesis in G₀/G₁ (d) and S/G₂/M (e) cells from Cy/G-CSF-treated mice (n=10 mice from 6 experiments). Extended Data Fig. 3f–g show the data from Fig. 3d–e side-by-side with data from untreated controls in Fig. 3b. f, Cell diameter (n>60 cells/population from 2 mice). g, 18S rRNA and 28S rRNA content in 15,000 cells from each stem/progenitor cell population (n=3 mice). All data represent mean±s.d. To assess the statistical significance of treatment effects within the same cells (b-c) we performed two-tailed Student’s t-tests (*, p<0.05; **, p<0.01; ***, p<0.001) and differences between HSCs and other cells (b-g) were assessed with a repeated measures one-way ANOVA followed by Dunnett’s test for multiple comparisons (#, p<0.05; ##, p<0.01; ###, p<0.001).

Figure 4. Rpl24^Bst/+ HSCs synthesize less protein and have less capacity to reconstitute irradiated mice

a, Frequencies of HSCs and MPPs in Rpl24^Bst/+ (n=7) versus littermate control (n=6) mice (n=5 experiments). b,c Frequency of HSCs in S/G₂/M (b; n=4 untreated mice/genotype, n=3 (Rpl24^Bst/+) or 4 (+/+) Cy/G-CSF treated mice/genotype in 3 experiments) and frequency of HSCs in the bone marrow (c; n=6 wild-type and 7 Rpl24^Bst/+ untreated mice, n=7 wild-type and 5 Rpl24^Bst/+ Cy/G-CSF treated mice in 4 experiments) after treatment with Cy/G-CSF. d, Protein synthesis in haematopoietic cells based on OP-Puro incorporation in vivo (n=4 mice/genotype in 4 experiments). e, Donor cell engraftment when 5×10⁵ donor bone marrow cells were transplanted along with 5×10⁵ recipient bone marrow cells into irradiated recipient mice (n=4 experiments with a total of 17 recipients for wild-type, and 20 for Rpl24^Bst/+; myeloid, B, and T engraftment are in Extended Data Figure 5k). f, The number of long-term multilineage reconstituted secondary recipients (>0.5% donor myeloid and lymphoid cells for at least 16 weeks after transplantation) after secondary transplantation of 3×10⁶ bone marrow cells from primary recipients in (e) (n=4 donors/genotype). All data represent mean±s.d. Statistical significance was assessed with two-tailed Student’s t-tests (a-e) and Fisher’s exact test (f) (*p<0.05, **p<0.01, ***p<0.001). Differences between HSCs and other cell populations (d) were assessed with a repeated measures one-way ANOVA followed by Dunnett’s test for multiple comparisons (#, p<0.05; ##, p<0.01; ###, p<0.001).

Figure 5. Rpl24^Bst/+ blocks the increase in protein synthesis and restores HSC function after Pten deletion

a, Western blots of 30,000 cells from each population. Long and short exposures for pS6 are shown. For total S6, a non-specific band is present below the specific band. Differences in β-Actin represent differences in β-Actin content per cell (1 representative blot from two experiments). b, Representative histograms of OP-Puro fluorescence in HSCs of each genotype. c, OP-Puro incorporation into HSCs of each genotype (n=15 experiments). d, Western blots of 30,000 HSCs/MPPs of the indicated genotypes (1 representative blot from two experiments). e, Representative spleens 2 weeks after pIpC administration to wild-type, Mx-1-Cre; Pten^fl/fl, Rpl24^Bst/+, and Mx-1-Cre; Pten^fl/fl; Rpl24^Bst/+ mice. f, Spleen and thymus cellularity (n=7 experiments). g, Time until mice had to be sacrificed due to illness after transplantation of 2×10⁶ bone marrow cells of the indicated genotypes into irradiated recipient mice. h, i, 10 donor HSCs were transplanted along with 3×10⁵ recipient bone marrow cells into irradiated recipients. Donor cell engraftment (h) and fraction of recipients that were long-term multilineage reconstituted (i; 3 experiments). j, k, Donor cell engraftment (j) and the fraction of secondary recipients that were long-term multilineage reconstituted (k) after transplantation of 3×10⁶ bone marrow cells from primary recipients in (h) (n=4 donors/genotype). All data represent mean±s.d. Differences among genotypes (c, f, h) were assessed with a one way ANOVA followed by Dunnett’s test for multiple comparisons. Statistical significance was assessed by log-rank test (g), Chi-squared tests followed by Tukey's t-tests for pairwise comparisons (i,k), or a one way ANOVA followed by Tukey’s t-tests for multiple comparisons (j). Significance was expressed relative to wild-type (#, p<0.05; ##, p<0.01; ###, p<0.001) or Pten-deficient cells (*, p<0.05; **, p<0.01; ***, p<0.001).