Thrombopoietin-induced expression of the glycoprotein IIb gene involves the transcription factor PU.1/Spi-1 in UT7-Mpl cells

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 Eb 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.