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 diffe rentiation of megakaryocytes and their progenitors. These actions can be reproduced in the human megakaryoblastic cell line UT7 into which t he murine TPO receptor, c-Mpl, was introduced, In these cells, TPO enh anced the expression of the specific megakaryocytic marker integrin gl ycoprotein (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 promote r fragments, we delineated a TPO-responsive element within the previou sly reported enhancer region of the promoter. Although this enhancer i ncluded GATA- and Ets-binding sites (EBSs), we found that only EBS -51 4 was important for TPO response, We identified PU.1/Spi-1 as the endo genous Ets transcription factor that strongly and preferentially inter acted 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 bindi ng activity in UT7-Mpl cells, in contrast, TPO did not affect the expr ession of Ets-1/2 while weakly increasing the levels of Fli-1. Overexp ression of PU.1/Spi-1 was further shown to enhance GPIIb promoter acti vity in the absence and presence of TPO. Overall, our data indicated t hat, in UT7-Mpl cells, TPO increased the transcriptional activity of a GPIIb gene in part due to an enhanced expression of an unexpected tra nscription 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.