Abstract

Thrombopoietin (Tpo) is the primary cytokine regulator of megakaryocytopoiesis. Tpo engagement of its receptor, Mpl, activates the classic MAP kinase (Raf/MEK/ERK) pathway, but the biological role of MAP kinase signaling in megakaryocytopoiesis remains poorly defined. Raf family kinases control signal flow through the classic MAP kinase pathway from activated cytokine receptors. We therefore undertook a genetic approach to understand the role of MAP kinase signaling and to identify which Raf family member is important for megakaryocytopoiesis. Using data from in vitro B-raf-/- ES cell cultures, fetal liver hematopoietic cells from mid-gestation B-raf-/- embryos, and B-raf-/- chimeric mice, we recently reported that B-Raf acts in a cell autonomous manner to quantitatively affect megakaryocytopoiesis. However, limitations of the chimeric mouse model, and mid-gestation lethality of B-raf-/- embryos precluded a detailed mechanistic understanding of B-Raf activity or the evaluation of B-Raf in adult megakaryocytopoiesis. We therefore generated tissue-restricted B-Raf deficient mice by crossing B-rafflox/flox mice with mice expressing Cre recombinase controlled by the Tie2 promoter/enhancer. The Tie2Cre+/B-rafflox/flox mice were born with normal Mendelian genetics and without gross abnormalities. Circulating leukocytes demonstrated complete recombination of the floxed B-Raf allele, and western blots showed undetectable B-Raf expression in platelet, spleen, and thymus lysates, consistent with complete hematopoietic Cre-mediated recombination. Steady state platelet counts were not altered in the B-Raf deficient animals at baseline: Tie2Cre+/B-rafflox/flox mice = 788 +/−57 x 103/mm3; Tie2Cre-/B-rafflox/flox mice 800 +/−40 x 103/mm3 (p=0.76, n=4). However, Tie2Cre+/B-rafflox/flox mice had a markedly impaired platelet count rise following Tpo injection, with peak counts of 3,375 +/−752 x 103/mm3 compared with 5,320 +/−606 x 103/mm3 for Tie2Cre-/B-rafflox/flox mice (p=0.0147, n=4) at 6 days post injection. In vitro expansion of CD41+ cells from Tie2Cre+/B-rafflox/flox bone marrow was only a third that of control mice, suggesting that the impaired in vivo platelet rise following Tpo reflects, at least in part, a decreased expansion of megakaryocyte lineage cells. Day 4 Tie2Cre+/B-rafflox/flox bone marrow cultures also yielded decreased low (2N-8N) and high (>32N) ploidy CD41+ cells compared with marrow from Tie2Cre-/B-rafflox/flox mice, while intermediate (16N-32N) ploidy CD41+ megakaryocytes were relatively preserved. TUNEL analysis revealed increased apoptotic death of the high ploidy (>32N) cells, a second possible mechanism contributing to the impaired platelet rise following Tpo injection. Together, our data demonstrate that the B-Raf/MAP kinase pathway is required for normal adult Tpo-induced thrombopoiesis through its effect on megakaryocyte lineage expansion and apoptotic cell death of mature megakaryocytes. Additional analysis is now underway to more fully define the role of B-Raf in megakaryocytopoiesis, including detailed biochemical studies to determine how the absence of B-Raf impacts intracellular signaling during this complex developmental process.

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