Establishment of model system for hematological disorders associated with mutated signal transduction molecules

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1. The signal transducer and activator of transcription 3 (Stat3), a member of the Stat family of proteins, is commonly activated by thrombopoietic cytokines including thrombopoietin (TPO), interleukin (IL)-6, and interleukin-11. This finding strongly suggested that Stat3 has an important role in megakaryopoiesis and thrombopoiesis. To clarify the functional role of Stat3 in in vivo megakaryopoiesis and thrombopoiesis, we generated transgenic mice overexpressing a dominant-negative Stat3, Stat3F, to suppress the function of endogenous Stat3. To accomplish the selective expression of Stat3F in megakaryocytic lineage cells, we used the regulatory gene region of GATA-1 transcription factor selectively expressed in megakaryocytic and erythroid lineage cells. Two independent transgenic (Tg) mice lines were established. Platelet counts were within the normal range in steady-state conditions and were recovered normally from transient thrombocytopenia induced by antiplatelet serum injection. I … More nterestingly, the platelet recovery from myelosuppression after 5-fluorouracil treatment was significantly delayed in the Tg mice. Collectively, our results strongly suggest that Stat3 plays an important role in the early stage of megakaryopoiesis, presumably through the expansion of megakaryocytic progenitor cells.2. FKHRL1, a member of the Forkhead transcription factor family, is a mammalian homologue of DAF-16 which plays an important role in the longevity of Caenorhabditis elegans. We found that FKHRL1 is one of the downstream molecules of phosphatidylinositol 3-kinase in the erythropoietin (EPO) signaling pathway, and that this molecule is phosphorylated directly by activated Akt, resulting in a loss of transcriptional activity. In addition, FKHRL1 mRNA was detectable in various kinds of hematopoietic cells including CD34+/CD38-, CD34+, glycophorin A+, glycoprotein II/IIIa+, and CD13+ cells. In particular, glycophorin A+ cells strongly expressed FKHRL1 at the mRNA level. To clarify the functional role of FKHRL1 in erythropoiesis, we prepared FKHRL1-TM which is replaced at three Akt phosphorylation sites, Thr32, Ser253, and Ser315 with alanine. Cord blood-derived CD34-positive cells were transduced with the bicistronic retroviral vector expressing FKHRL1-TM and green fluorescent protein (GFP) using an internal ribosome entry site (IRES). The amount of erythroid colony derived from FKHRL1-TM-transduced CD34+ cells was about 40% of that from the control vector-transduced cells. To elucidate the mechanism by which FKHRL1 inhibits erythroid colony formation, we generated a tamoxifen-inducible FKHRL1-TM : ER system in an EPO-dependent cell line, UT-7/EPO. Addition of tamoxifen inhibited the cell cycle growth and cell cycle progression. Our results suggest that FKHRL1 controls the cell cycling of erythroid cells via regulating cell cycle-associated gene expression. Less