Incomplete restoration of Mpl expression in the mpl-/- mouse produces partial correction of the stem cell-repopulating defect and paradoxical thrombocytosis

Abstract

Expression of Mpl is restricted to hematopoietic cells in the megakaryocyte lineage and to undifferentiated progenitors, where it initiates critical cell survival and proliferation signals after stimulation by its ligand, thrombopoietin (TPO). As a result, a deficiency in Mpl function in patients with congenital amegakaryocytic thrombocytopenia (CAMT) and in mpl(-/-) mice produces profound thrombocytopenia and a severe stem cell-repopulating defect. Gene therapy has the potential to correct the hematopoietic defects of CAMT by ectopic gene expression that restores normal Mpl receptor activity. We rescued the mpl(-/-) mouse with a transgenic vector expressing mpl from the promoter elements of the 2-kb region of DNA just proximal to the natural gene start site. Transgene rescued mice exhibit thrombocytosis but only partial correction of the stem cell defect. Furthermore, they show very low-level expression of Mpl on platelets and megakaryocytes, and the transgene-rescued megakaryocytes exhibit diminished TPO-dependent kinase phosphorylation and reduced platelet production in bone marrow chimeras. Thrombocytosis is an unexpected consequence of reduced Mpl expression and activity. However, impaired TPO homeostasis in the transgene-rescued mice produces elevated plasma TPO levels, which serves as an unchecked stimulus to drive the observed excessive megakaryocytopoiesis.

Figure 1

Transgene copy number and increased marrow megakaryocytosis in the Tg(mpl) mouse. (A) Southern blot determination of transgene copy number in DNA isolated from the marrows of Tg(mpl) mice and copy number standards generated by dilution of plasmid containing the mpl transgene into genomic DNA from the marrows of mpl^−/− mice. Transgene copy number in Tg(mpl) mice was determined to be 38 based on band intensities quantitated by PhosphorImager. (B) Hematoxylin-eosin–stained sections of femurs from WT (C57BL/6J), mpl^−/−, and Tg(mpl) mice were examined by light microscopy (original magnification, × 200). (C) Morphologically recognizable megakaryocytes were counted in whole femur sections from each strain. Numbers represent the mean plus or minus SE of 6 femurs taken from 3 mice in each group. *P ≤ .003, Tg(mpl) versus WT and Tg(mpl) versus mpl^−/−. (D) Representative 2-color flow cytometric analysis of indirect immunofluorescence staining of 8- to 10-week-old WT, mpl^−/−, and Tg(mpl) mice bone marrow populations stained with anti-CD41–FITC, anti-CD14–PE, and isotype-matched control antibodies. The percentage of cells staining CD41⁺/CD14⁻ is shown in the bottom right of each histogram and is representative of 3 independent experiments (4 mice from each strain were pooled in each experiment).

Figure 2

Comparison of hematopoietic progenitors in the marrows of WT, mpl^−/−, and Tg(mpl) mice. (A) BFU-E, CFU-GM, CFU-GEMM, and CFU-Mk progenitors were grown in methylcellulose and collagen–based media and enumerated; + indicates no CFU-GEMM colonies grew from mpl^−/− marrow; n = 3 mice from each strain. *P = .006 versus mpl^−/−; **P ≤ .013 for WT versus mpl^−/− and for Tg(mpl) versus mpl^−/−; ***P ≤ .014 for WT versus mpl^−/− and for Tg(mpl) versus mpl^−/−. (B) Size of CFU-Mk colonies. The CFU-Mk colonies (total counts shown in panel A) were further characterized by size. There were significantly fewer large (> 20-cell) colonies in Tg(mpl) than WT marrow (P < .005). Means plus or minus SE are shown.

Figure 3

Hematopoietic stem cell repopulating ability of WT, mpl^−/−, and Tg(mpl) marrow. WT (Ly5.2), mpl^−/−, and Tg(mpl) marrow competed 1:1 with WT (Ly5.1) marrow cells in lethally irradiated WT (Ly5.1) recipients. The repopulating activity was determined at 16 weeks after transplantation from the percentage of peripheral blood leukocytes in reconstituted chimeras staining for each of the CD45 alloantigens, CD45.1 and CD45.2. Repopulating activity is normalized to the WT (Ly5.1) control marrow; n = 8 to 9 mice for each strain. Means plus or minus SE are shown.

Figure 4

Plasma TPO levels and platelet Mpl expression. (A) TPO levels determined by ELISA on peripheral blood plasma from WT, mpl^−/−, and Tg(mpl) mice; n = 3 to 4 mice for each strain. *P ≤ .003, Tg(mpl) versus WT and Tg(mpl) versus mpl^−/−. Means plus or minus SE are shown. (B) Expression of Mpl on platelet lysates analyzed by Western blot probed with Mpl antiserum (top panel). The blot was stripped and reprobed with anti–pan-actin antibody to ensure equal amount of protein in each lane (bottom panel). Levels of Mpl in Tg(mpl) platelets are 9-fold lower than that in WT.

Figure 5

WBC and platelet reconstitution in chimeric mice. (A) Irradiated Tg(mpl) recipients were reconstituted with a 1:3 ratio of marrow cells from Tg(GFP) and Tg(mpl) donors. At 16 weeks after transplantation, the contribution of peripheral blood leukocytes and platelets coming from each of the engrafted donor marrow stem cells in chimeric mice was determined by flow cytometry; n = 15 mice. (B) A comparison of platelet counts and TPO levels in mice that received transplants that resulted in bone marrow chimeras (n = 15) and from mice that were reconstituted with only Tg(mpl) stem cells (n = 4). Means plus or minus SE are shown.

Figure 6

TPO-induced signaling in WT and Tg(mpl) megakaryocytes. Bone marrow cells were harvested from mice and grown in serum-free medium with 37.5 ng/mL mTPO for 3 days; megakaryocytes were then purified using an albumin density-gradient column achieving a final enrichment of greater than 90%. (A) Western blots using Mpl antiserum demonstrates the relative Mpl expression in both WT and Tg(mpl) megakaryocytes. (B) Isolated megakaryocytes were incubated in a serum-free, cytokine-free medium for 10 hours and then stimulated with 50 ng/mL or 500 ng/mL mTpo for 12 minutes. Total cell lysates, normalized for protein concentration, were separated by 4% to 20% SDS-PAGE and immunoblotted with the indicated phosphospecific antibodies (Jak2, Stat3, Stat5, and MAPK; “Methods”). Blots were then stripped and reprobed with appropriate antibodies to ensure equal amounts of protein in each lane (bottom panels). Relative phosphorylation levels were quantitated by densitometry. Results are representative of 2 independent experiments.

Figure 7

Expression levels of mpl in megakaryocytes and LSK cells. Expression levels of mpl mRNA, normalized to Gapdh levels, was determined in megakaryocytes and LSK cells from WT and Tg(mpl) mice. Results shown were compiled from 3 separate experiments using pooled samples from 3 mice in each strain per experiment. Means plus or minus SE are shown.