Doubeikovski A, Uzan G, Doubeikovski Z, Prandini M H, Porteu F, Gisselbrecht S, Dusanter-Fourt I
INSERM U363, Institut Cochin de Génétique Moléculaire, Hopital Cochin, 27 rue du Faubourg Saint-Jacques, 75014 Paris, France.
J Biol Chem. 1997 Sep 26;272(39):24300-7. doi: 10.1074/jbc.272.39.24300.
Thrombopoietin (TPO) is the major regulator of proliferation and differentiation of megakaryocytes and their progenitors. These actions can be reproduced in the human megakaryoblastic cell line UT7 into which the murine TPO receptor, c-Mpl, was introduced. In these cells, TPO enhanced the expression of the specific megakaryocytic marker integrin glycoprotein (GP) IIb-IIIa while decreasing the expression of erythroid genes (Porteu, F., Rouyez, M. -C., Cocault, L., Benit, L., Charon, M., Picard, F., Gisselbrecht, S. , Souyri, M., and Dusanter-Fourt, I. (1996) Mol. Cell. Biol. 16, 2473-2482). We have now analyzed the effect of TPO on the transcriptional activity of the GPIIb promoter in these cells. Using transient transfection assays of a series of human GPIIb promoter fragments, we delineated a TPO-responsive element within the previously reported enhancer region of the promoter. Although this enhancer included GATA- and Ets-binding sites (EBSs), we found that only EBS -514 was important for TPO response. We identified PU. 1/Spi-1 as the endogenous Ets transcription factor that strongly and preferentially interacted with this enhancer EBS. This factor did not interact with other proximal EBSs in the GPIIb promoter. We next showed that TPO induced a strong and selective increase of PU. 1/Spi-1 expression and DNA binding activity in UT7-Mpl cells. In contrast, TPO did not affect the expression of Ets-1/2 while weakly increasing the levels of Fli-1. Overexpression of PU.1/Spi-1 was further shown to enhance GPIIb promoter activity in the absence and presence of TPO. Overall, our data indicated that, in UT7-Mpl cells, TPO increased the transcriptional activity of a GPIIb gene in part due to an enhanced expression of an unexpected transcription factor, the Ets family PU.1/Spi-1 factor. To our knowledge, this is the first evidence of a role for the PU.1/Spi-1 factor in the regulation of megakaryocytic genes.
血小板生成素（TPO）是巨核细胞及其祖细胞增殖和分化的主要调节因子。这些作用可在导入了小鼠TPO受体c-Mpl的人巨核母细胞系UT7中重现。在这些细胞中，TPO增强了特异性巨核细胞标志物整合素糖蛋白（GP）IIb-IIIa的表达，同时降低了红系基因的表达（波特厄，F.，鲁耶，M.-C.，科考，L.，贝尼特，L.，沙龙，M.，皮卡德，F.，吉塞尔布雷希特，S.，苏伊里，M.，以及迪桑特-富尔，I.（1996年）《分子与细胞生物学》16，2473 - 2482）。我们现在分析了TPO对这些细胞中GPIIb启动子转录活性的影响。通过对一系列人GPIIb启动子片段进行瞬时转染分析，我们在启动子先前报道的增强子区域内划定了一个TPO反应元件。尽管这个增强子包含GATA结合位点和Ets结合位点（EBSs），但我们发现只有EBS -514对TPO反应很重要。我们鉴定出PU.1/Spi-1是与该增强子EBS强烈且优先相互作用的内源性Ets转录因子。该因子不与GPIIb启动子中的其他近端EBS相互作用。接下来我们表明，TPO在UT7-Mpl细胞中诱导了PU.1/Spi-1表达和DNA结合活性的强烈且选择性增加。相比之下，TPO不影响Ets-1/2的表达，而只是微弱增加Fli-1的水平。进一步表明，在不存在和存在TPO的情况下，PU.1/Spi-1的过表达均能增强GPIIb启动子活性。总体而言，我们的数据表明，在UT7-Mpl细胞中，TPO部分通过增强一种意外转录因子——Ets家族的PU.1/Spi-1因子的表达，增加了GPIIb基因的转录活性。据我们所知，这是PU.1/Spi-1因子在调节巨核细胞基因中发挥作用的首个证据。
