Mpl expression on megakaryocytes and platelets is dispensable for thrombopoiesis but essential to prevent myeloproliferation

Abstract

Thrombopoietin (TPO) acting via its receptor, the cellular homologue of the myeloproliferative leukemia virus oncogene (Mpl), is the major cytokine regulator of platelet number. To precisely define the role of specific hematopoietic cells in TPO-dependent hematopoiesis, we generated mice that express the Mpl receptor normally on stem/progenitor cells but lack expression on megakaryocytes and platelets (Mpl(PF4cre/PF4cre)). Mpl(PF4cre/PF4cre) mice displayed profound megakaryocytosis and thrombocytosis with a remarkable expansion of megakaryocyte-committed and multipotential progenitor cells, the latter displaying biological responses and a gene expression signature indicative of chronic TPO overstimulation as the underlying causative mechanism, despite a normal circulating TPO level. Thus, TPO signaling in megakaryocytes is dispensable for platelet production; its key role in control of platelet number is via generation and stimulation of the bipotential megakaryocyte precursors. Nevertheless, Mpl expression on megakaryocytes and platelets is essential to prevent megakaryocytosis and myeloproliferation by restricting the amount of TPO available to stimulate the production of megakaryocytes from the progenitor cell pool.

Keywords: bone marrow; essential thrombocythemia.

Fig. 1.

Targeted modification of the Mpl locus in mice and Mpl expression in Mpl^{PF4cre/PF4cre} mice. (A) Generation of a GFP reporter allele of Mpl expression that also allows conditional, cre-mediated Mpl inactivation. Mpl⁺, wild type Mpl allele. Exons 7–12 (filled boxes), 3′ untranslated region of exon 12 (striped). Mpl^T, targeting vector incorporated into the Mpl locus. IRES-GFP cassette (white), PGK-neo selection cassette (shaded). Mpl^fl, GFP reporter for expression from the Mpl locus. PGK-neo excised by intercrossing with Flp recombinase transgenic mice. Mpl^cre, Mpl null allele generated via cre-mediated excision. (B) Western blot of protein extracted from platelets (100 μg) and megakaryocytes (Megs, 188 μg) pooled from two to three independent genotype-matched Mpl^fl/fl and Mpl^{PF4cre/PF4cre} mice per lane. (C) The mean fluorescence intensity of Mpl expression on stem and progenitor cell populations from Mpl^fl/fl, Mpl^{PF4cre/PF4cre} and Mpl^−/− mice is shown relative to the isotype control (SI Appendix, Fig. S3C). Mean and SD shown, n = 4 mice per genotype. *P < 0.005 by two-tailed Student t test. (D) Transcriptional activity of the Mpl locus via expression of the IRES-GFP cassette in stem and progenitor populations. GFP mean fluorescence intensity from Mpl^fl/fl and Mpl^{PF4cre/PF4cre} cells is shown relative to Mpl^{Delcre/Delcre} mice (SI Appendix, Fig. S3D). Mean and SD shown, n = 3–8 mice per genotype. *P < 0.002.

Fig. 2.

Expanded megakaryopoiesis in Mpl^{PF4cre/PF4cre} mice. (A) Number of megakaryocytes per high powered field from bone marrow (Left) and spleen (Right) of Mpl^+/+, Mpl^fl/fl, Mpl^{PF4cre/PF4cre}, and TPO^Tg mice. *P < 0.03 by Student's unpaired two-tailed t test, n = 3–9 mice per genotype. (B) Ploidy of bone marrow megakaryocytes from Mpl^fl/fl, Mpl^{PF4cre/PF4cre}, and TPO^Tg mice. Mean and SD shown, n = 4–6 mice per genotype. *P_adj < 0.03 by Student's unpaired two-tailed t test using Bonferroni testing for multiple comparisons.

Fig. 3.

Expanded progenitors in Mpl^{PF4cre/PF4cre} and TPO^Tg mice. Numbers of cells in flow cytometrically defined bone marrow stem and progenitor cell fractions from Mpl^fl/fl (n = 8), Mpl^{PF4cre/PF4cre} (n = 6), Mpl^{Delcre/Delcre} (n = 3), and TPO^Tg (n = 5) mice shown as cells per femur relative to Mpl^+/+ (n = 4) controls. For definitions of cell populations, see SI Appendix, Fig. S5 and Table S1. Mean and SD shown. *P_adj < 0.05 by two-tailed Student's unpaired t test compared with Mpl^+/+; θP_adj < 0.05 by two-tailed Student's unpaired t test specifically for Mpl^{PF4cre/PF4cre} compared with Mpl^fl/fl with Bonferroni testing for multiple comparisons.

Fig. 4.

TPO transcription, circulating TPO concentration, and gene expression changes in LSK cells of Mpl^{PF4cre/PF4cre} mice. (A) TPO concentration by immunoassay in serum from Mpl^+/+, Mpl^fl/fl, Mpl^{PF4cre/PF4cre}, Mpl^−/−, and TPO^Tg mice. *P < 0.04, n = 10–17 mice per genotype. (B) TPO expression in livers determined by quantitative RT-PCR with ΔCT shown as mean and SD relative to GAPDH expression. There were no significant differences among Mpl^+/+, Mpl^fl/fl, and Mpl^{PF4cre/PF4cre} genotypes, n = 3–5 mice per group. (C) Barcode plot showing ability of TPO^Tg “TPO stimulated” LSK signature genes and Mpl^−/− “TPO starved” signature genes (SI Appendix, Tables S3 and S4) to distinguish between Mpl^{PF4cre/PF4cre} LSKs and Mpl^fl/fl LSKs, with corresponding P values derived from rotational gene set testing using ROAST. Red bars designate up-regulated genes in the TPO^Tg TPO stimulated LSK signature set (P = 2 × 10⁻⁴); blue bars designate Mpl^{fl/fl −/−} TPO starved LSK signature set (P = 8 × 10⁻⁴); demonstrating gene expression in Mpl^{PF4cre/PF4cre} LSKs is strongly correlated with a TPO stimulated gene signature. (D) Barcode plot showing the TPO stimulated gene expression signature of Mpl^{PF4cre/PF4cre} LSKs (red bars, up-regulated genes; blue bars, down-regulated genes; SI Appendix, Table S2) can distinguish between CD34⁺ bone marrow cells of patients with essential thromboocythemia from controls (ref. ; GEO database accession no. GSE9827, P = 1.48 × 10⁻²). P value derived from rotational gene set testing by using ROAST with the Mpl^{PF4cre/PF4cre} LSK gene signature weighted by log fold-change (SI Appendix, SI Materials and Methods, Table S2, and Fig. S7).

Fig. 5.

Model for regulation of TPO and control of megakaryopoiesis. (Upper) Clearance of TPO by Mpl-expressing megakaryocytes in bone marrow and the peripheral blood platelet pool maintains TPO homeostasis at steady state and in situations of acute thrombocytopenia. For definitions of cell populations, see SI Appendix, Table S1. Mpl expression is shown as blue bars for each population in Mpl^fl/fl mice. (Lower) Loss of TPO clearance by megakaryocytes and platelets leads to excessive TPO stimulation of Mpl-expressing HSCs and progenitor cells, multilineage progenitor expansion, and differentiation toward the megakaryocyte lineage from bipotential megakaryocyte-erythroid progenitors, with consequent myeloproliferation, megakaryocytosis, and thrombocytosis. Whereas the availability of TPO for stimulation of stem/progenitor cells is increased, consumption by the expanded numbers of these cells normalizes circulating TPO concentration. PF4-cre expression is shown as green bars and Mpl expression is shown as blue bars for each population in Mpl^{PF4cre/PF4cre} mice.