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

1. 信号换能器和转录激活因子3 （Stat3）是Stat蛋白家族的一员，通常被血小板生成细胞因子激活，包括血小板生成素（TPO）、白细胞介素(IL)-6和白细胞介素-11。这一发现强烈提示Stat3在巨核生成和血小板生成中具有重要作用。为了阐明Stat3在体内巨核生成和血小板生成中的功能作用，我们培养了过表达Stat3显性阴性基因Stat3F的转基因小鼠，以抑制内源性Stat3的功能。为了实现Stat3F在巨核细胞系细胞中的选择性表达，我们利用GATA-1转录因子调控基因区在巨核细胞系和红系细胞中选择性表达。建立了两个独立的转基因（Tg）小鼠系。在稳态条件下，血小板计数在正常范围内，抗血小板血清注射引起的短暂性血小板减少症恢复正常。更有趣的是，在Tg小鼠中，5-氟尿嘧啶治疗后骨髓抑制的血小板恢复明显延迟。总的来说，我们的结果强烈表明Stat3在巨核细胞形成的早期阶段发挥重要作用，可能是通过巨核细胞祖细胞的扩增。FKHRL1是叉头转录因子家族的成员，是DAF-16的哺乳动物同源物，DAF-16在秀丽隐杆线虫的长寿中起重要作用。我们发现FKHRL1是促红细胞生成素（EPO）信号通路中磷脂酰肌醇3-激酶的下游分子之一，该分子被活化的Akt直接磷酸化，导致转录活性丧失。此外，FKHRL1 mRNA在CD34+/CD38-、CD34+、糖蛋白A+、糖蛋白II/IIIa+、CD13+等多种造血细胞中均可检测到。特别是，糖蛋白A+细胞在mRNA水平上强烈表达FKHRL1。为了阐明FKHRL1在红细胞生成中的功能作用，我们制备了FKHRL1- tm，它在Akt的三个磷酸化位点Thr32、Ser253和Ser315上被丙氨酸取代。脐带血来源的cd34阳性细胞通过内部核糖体进入位点（IRES）被表达FKHRL1-TM和绿色荧光蛋白（GFP）的双链逆转录病毒载体转导。fkhrl1 - tm转导的CD34+细胞获得的红系集落数量约为对照载体转导细胞的40%。为了阐明FKHRL1抑制红系集落形成的机制，我们在EPO依赖细胞系UT-7/EPO中构建了他莫昔芬诱导的FKHRL1- tm: ER系统。他莫昔芬的加入抑制了细胞周期生长和细胞周期进程。我们的研究结果表明，FKHRL1通过调节细胞周期相关基因的表达来控制红细胞的细胞周期。少

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