Signals emanating from the membrane proximal region of the thrombopoietin receptor (mpl) support hematopoietic stem cell self-renewal

Abstract

Objective: Studies using thrombopoietin -/- (TPO(-/-)) or TPO receptor, mpl(-/-) mice have established a critical role for TPO/mpl signaling in hematopoietic stem cell (HSC) development. In this study, we further dissected mpl signaling in both megakaryopoiesis and HSC function, using mice bearing a truncated mpl receptor lacking the distal 60 amino acids (Delta60). This deletion removes three major signaling tyrosines on the mpl cytoplasmic domain, but retains the membrane proximal Box1 and Box2 domains required for JAK2 activation.

Materials and methods: Competitive bone marrow transplantations (BMT) and serial BMTs were performed to study HSC function. Western blot analysis was used to study TPO-stimulated signaling pathways. BM cell cultures in the presence of TPO were used to study megakaryocyte development.

Results: In agreement with prior findings, we show that Delta60 BM cells cultured in TPO generated normal numbers of megakaryocytes, but with greatly reduced ploidy. As expected from the deletion of three signaling tyrosine residues, freshly isolated Delta60 megakaryocytes showed marked reduction in all known TPO-stimulated signaling pathways tested, including signal transducers and activators of transcription (Stat) 5, Stat3, Akt, and p42/44 mitogen-activated kinase. We found that Delta60 mice displayed normal short-term (ST-HSC) activities and marginally compromised long-term (LT-HSC) stem cell activities in primary transplantation. In addition, Delta60 mice supported HSC self-renewal for at least two serial BMTs.

Conclusion: Our data reveal a pivotal role for an unknown signal emanating from the membrane proximal region of the mpl receptor or from JAK2 itself in maintaining stem cell activity and self-renewal, in addition to its role in megakaryocytopoiesis and thrombopoiesis.

Figure 1. Δ60 mice expressed comparable levels of surface mpl in both megakaryocytes and stem cells similarly to WT controls

(a) CD41⁺ BM cells from WT, Δ60, or mpl-/- mice were first enriched using magnetic bead selection. Subsequently, the cells were stained for PE-mpl. Percentages of CD41⁺mpl^- cells (lower right box) and CD41⁺mpl⁺ cells (upper right box) are shown in each plot. (b) Lin^- BM cells from WT, Δ60, or mpl-/- mice were isolated using magnetic bead purification, and stained for Sca-1, Kit, and mpl. Percentages of Lin^- cells double positive for Sca-1 and Kit are shown in the upper panels. Median fluorescence levels of PE-mpl are shown in the lower panels for Lin^-Sca1⁺Kit⁺ (LSK) cells.

Figure 1. Δ60 mice expressed comparable levels of surface mpl in both megakaryocytes and stem cells similarly to WT controls

(a) CD41⁺ BM cells from WT, Δ60, or mpl-/- mice were first enriched using magnetic bead selection. Subsequently, the cells were stained for PE-mpl. Percentages of CD41⁺mpl^- cells (lower right box) and CD41⁺mpl⁺ cells (upper right box) are shown in each plot. (b) Lin^- BM cells from WT, Δ60, or mpl-/- mice were isolated using magnetic bead purification, and stained for Sca-1, Kit, and mpl. Percentages of Lin^- cells double positive for Sca-1 and Kit are shown in the upper panels. Median fluorescence levels of PE-mpl are shown in the lower panels for Lin^-Sca1⁺Kit⁺ (LSK) cells.

Figure 2. Δ60 megakaryocytes showed reduced TPO-mediated differentiation during in vitro megakaryocyte culture

Total BM cells from WT mice or Δ60 mice were cultured in different concentrations of TPO for 4 days, (a) the numbers of CD41⁺ megakaryocytes with 8N and greater ploidy were enumerated (mean ± SD). (b) Mean ploidies (±SD) of BM-derived megakaryocytes were plotted.

Figure 3. Markedly reduced TPO-stimulated signaling pathways in Δ60 megakaryocytes

Freshly- isolated CD41⁺ BM megakaryocytes from WT or Δ60 mice were stimulated with 0, 10 or 50 ng/ml of TPO for 10 mins. Protein lysates from equal number of cells were subjected to Western Blotting analysis. Phosphorylation and total protein levels of Stat5 (a), Stat3 (b), p42/44MAPK and Akt (c), are shown.

Figure 4. Δ60 mice showed slight but significant decrease in stem cell repopulating capability four months after transplantation

BM cells from WT, Δ60, and mpl-/- mice were mixed at 1:1, 3:1, or 9:1 ratio with competitor cells, and transplanted into irradiated recipient mice. Chimerism of transplanted mice were measured 4 months after transplantation. Five mice per genotype per group were used in each experiment and the results were pooled from three independent experiments. Students’ t-tests were carried out and the p values for the differences between Δ60 or mpl-/- mice and WT mice are shown on the top of each bar (n=15).

Figure 5. Δ60 mice showed decreased stem cell maintenance eight months after transplantation

BM cells from WT, Δ60, and mpl-/- mice were mixed at 1:1 or 9:1 ratio with Ly5.1 competitor cells, and transplanted into irradiated recipient mice. Chimerism of transplanted mice were measured 1, 4, and 8 months after transplantation. (a) shows the outcome of the 1:1 test to competitor ratio, and (b) shows that of the 9:1 test to competitor ratio. Five mice per genotype were used in each experiment, and the results were pooled from three independent experiments. Students’ t-tests were carried out and the p values of Δ60 comparing to WT are shown on top of each point (n=15).

Figure 5. Δ60 mice showed decreased stem cell maintenance eight months after transplantation

BM cells from WT, Δ60, and mpl-/- mice were mixed at 1:1 or 9:1 ratio with Ly5.1 competitor cells, and transplanted into irradiated recipient mice. Chimerism of transplanted mice were measured 1, 4, and 8 months after transplantation. (a) shows the outcome of the 1:1 test to competitor ratio, and (b) shows that of the 9:1 test to competitor ratio. Five mice per genotype were used in each experiment, and the results were pooled from three independent experiments. Students’ t-tests were carried out and the p values of Δ60 comparing to WT are shown on top of each point (n=15).

Figure 6. Δ60 mice show moderate defects in supporting stem cell self-renewal

(a) Primary BM transplanted mice with 3:1 test to competitor ratio were sacrificed at four months, and 5 million total BM cells were transplanted into each secondary recipient mouse. Chimerism of the secondary transplanted mice was measured after four months. In each experiment, five mice were used in each primary and secondary transplant, and the results were pooled from three independent experiments (n=15). (b) In one experiment, the secondary transplanted mice were continued for tertiary and quaternary transplants. 20 million total BM cells were used in each tertiary and quaternary recipient mouse. Chimerism of tertiary and quaternary transplanted mice were measured at four to five months post trasnsplant (n=10). Students’ t-tests were performed and the p values of Δ60 compared to WT are shown. Since the donor cells from mpl-/- mice did not show any reconstitution at the secondary and tertiary transplants, this group was terminated after the tertiary BMT.