Phosphatidylinositol 3-Kinase Is Necessary but Not Sufficient for Thrombopoietin-induced Proliferation in Engineered Mpl-bearing Cell Lines as Well as in Primary Megakaryocytic Progenitors

Abstract

Mature megakaryocytes are large, polyploid bone marrow cells that give rise to circulating platelets (1). Thrombopoietin (TPO) 1 has been characterized as the primary hematopoietic cytokine regulating normal megakaryocyte (MK) development. The receptor for TPO is encoded by the c-mpl proto-oncogene, a member of the hematopoietic growth factor receptor family, which in altered form causes a myeloproliferative syndrome in mice (2). In addition to its effects on MK development, TPO has been found to play a significant role in promoting stem cell survival, and in conjunction with other cytokines, it can support stem cell expansion (3-5). Characterization of the pathways by which TPO signals to promote survival and proliferation in stem cells and developing MKs is critical to a better understanding of the physiologic, pathologic, and potentially therapeutic roles of the cytokine in stem cell expansion, myeloproliferative disorders, and states of bone marrow failure.Many of the effects of TPO on cell survival and proliferation have been ascribed to activation of the Jak/STAT and Ras/Raf/ MAPK pathways (reviewed in Ref. 6). Activation of Jak2 leads to tyrosine phosphorylation of Mpl as well as STAT3 and STAT5 (7,8), and the phosphorylated STATs dimerize and translocate to the nucleus, where they stimulate transcription. In addition, phosphorylation of distal tyrosine residues on Mpl creates docking sites for SHC, which undergoes phosphorylation and then recruits Grb2/SOS, thus activating the Ras/Raf/ MAPK pathway (8 -10). Activation of the Ras/Raf/MAPK pathway can also occur by an alternative, as yet undetermined mechanism, independent of the distal receptor phosphotyrosine residues (11).Although these studies and others have shown that activation of the Jak/STAT and Ras/Raf/MAPK pathways is important for signaling by Mpl, other pathways likely contribute to the cellular response to TPO. One such pathway that may contribute to Mpl signaling is phosphatidylinositol 3-kinase (PI3K). PI3K has been shown to be activated by many growth factors involved in hematopoiesis, including stem cell factor, platelet-derived growth factor, erythropoietin, interleukin 3 (IL-3), IL-2, granulocyte-macrophage colony-stimulating factor, granulocyte colony stimulating factor, insulin-like growth factor 1, and TPO (12-16). It has been well established that activation of PI3K plays an important role in promoting cell survival (17-19). However, although there are ample data supporting a requirement for PI3K in proliferation of nonhematopoietic cells (20 -22), data showing specific requirements for PI3K in the cytokine-dependent proliferation of hematopoietic cells are inconclusive. Studies in cell lines with IL-2, IL-3, and erythropoietin have supported a role for PI3K in the proliferative response (23-26); however, studies with Flt-3L have not (27). Importantly, the conclusions from studies performed in primary cells often differ from those performed in cell lines. For example, data from cell lines support the role of PI3K in erythropoietin-...

Phosphoinositide 3-kinase/Akt signaling pathway and its therapeutical implications for human acute myeloid leukemia

“…23,71,121,204 However, it should not been overlooked that many cytokines activate PI3K/Akt signaling which therefore would seem to play an important role in erythropoiesis, myelopoiesis and thrombocytopoiesis. [244][245][246][247] Clearly, these results have been obtained in vitro and they might not reflect what happens in vivo. In addition, also in this field the findings are conflicting, as exemplified by a recent paper which suggests that EPO downregulates PI3K/Akt axis during erythropoiesis through upregulation of phosphatidylinositol 4-phosphatase II, 248 whereas other reports have suggested a key role for PI3K/Akt in EPO-mediated erythroid differentiation.…”

Increased megakaryocytopoiesis in Lyn-deficient mice

“…Both kinases and phosphatases play imperative roles in signaling through surface receptors by regulating the state of cellular phosphorylation. In addition to the Jak/ STAT signaling pathway, TPO has been shown to stimulate Ras/Raf/MAPK, and PI3K/Akt, affecting cell survival, growth, and differentiation Rojnuckarin et al, 1999;Geddis et al, 2001). Studies have now shown that the Src family of tyrosine kinases (SFKs) are activated and are involved in TPO signaling networks (Santini et al, 2002;Lannutti et al, 2003;Lannutti and Drachman, 2004).…”

“…MKs cell differentiation and proliferation are regulated by a number of cytokines; however, thrombopoietin (TPO) is the primary regulator of MK maturation including cellular enlargement and nuclear polyploidy. TPO binding to its cognate receptor, Mpl, results in the activation of a variety of signaling events including the Janus kinase (JAK)/signal transducer and activator of transcription (STAT), ras/raf/MAP kinase, and PI-3 kinase/Akt pathways Rojnuckarin et al, 1999;Geddis et al, 2001). Although Mpl lacks intrinsic kinase activity, dimerization of the receptor results in a rapid increase in tyrosine phosphorylation, in large part due to the transphosphorylation and activation of JAK2 Drachman et al, 1999).…”

The Roles of Phosphatidylinositol 3-Kinase and Protein Kinase Cζ for Thrombopoietin-induced Mitogen-activated Protein Kinase Activation in Primary Murine Megakaryocytes

“…Furthermore, by itself, PI3K is critical for cellular proliferation and survival (31) and may thus provide essential signals for the growth of cells containing truncated receptors. Akt, a downstream effector of PI3K, is phosphorylated in response to TPO in BaF3/T69 cells, suggesting that the PI3K pathway can be activated by the truncated receptor.…”

“…Our group has shown that PI3K is essential for cellular proliferation in BaF3/Mpl and primary MK (31). As TPO-induced Akt activation is much weaker in BaF3/T69 cells than in cells with full-length Mpl, its physiological relevance needed to be determined.…”