Thrombopoietin regulates IEX-1 gene expression through ERK-induced AML1 phosphorylation

The extracellular signal-regulated kinases (ERKs) are required for thrombopoietin (TPO) functions on hematopoietic cells, but the ERKS targets involved remain unknown. Here we show that the regulation of the immediate early gene X-1 (IEX-1), identified as an ERK substrate in response to TPO, was mediated by an ERK-dependent phosphorylation of AML1. The addition of TPO to UT7-Mpl cells and primary megakaryocytes induced gene expression of IEX-1. Neither erythropoietin (EPO) nor granulocyte macrophage-colony stimulating factor (GM-CSF) was able to activate IEX-1 gene expression in UT7-Mpl cells. The induced expression was mediated by a transcriptional activation of the IEX-1 promoter and required an AML1-binding site located at -1068. The direct involvement of AML1 in the regulation of IEX-1 gene expression was shown by both the use of AML1 mutants and by shRNA experiments targeting endogenous AML1. Finally, the ability of TPO to induce the IEX-1 gene expression was inhibited by U0126, a specific inhibitor of the ERKs activator MEK and AML1 transcriptional activity was shown to be modulated by TPO through ERK-dependent phosphorylation. Taken together, these data suggest that AML1 plays a role in modulating the IEX-1 expression and that the ERK-dependent AML1 phosphorylation regulates the TPO-mediated activation of IEX-1.