GATA-1, Transcriptionally, and Thrombopoietin-Mediated Akt Activation, Post-Translationally, Regulate Continuous Expression of Bcl-xL Protein during Megakaryopoiesis.

Abstract

Thrombopoietin (TPO) plays a relevant role for megakaryocyte differentiation from stem cells. One of the important biological activities of TPO is to prevent the apoptosis of megakaryocytic cells. As an anti-apoptotic protein Bcl-xL, which has been proved to be indispensable for erythroid differentiation, is also abundantly expressed in megakaryocytes, it is assumed that Bcl-xL plays an important role for megakaryopoiesis. We thus investigated the expression of Bcl-xL during megakaryopoiesis and the underlying regulatory mechanism. In stem cell-derived megakaryocytes, expression of Bcl-xL increased in the early- and mid-stages of the differentiation. Both in vitro in stem cell-derived megakaryocyteic cell culture and in vivo in an animal model injected with anti-platelet antibody, expression of Bcl-xL protein was maintained until platelet-producing stage of the megakaryopoiesis. TPO-depletion caused significant decrease in Bcl-xL protein level without affecting its mRNA in both stem cell-derived megakaryocytes and TPO-dependent megakaryocytic UT7/TPO cells. As a 12-kD fragment of Bcl-xL appeared by the withdrawal of TPO, we considered that Bcl-xL was cleaved upon TPO-depletion. This cleavage was blocked by a caspase-3-specific inhibitor, suggesting that caspase cleaves Bcl-xL in TPO-depleted megakaryocytes. Furthermore, pretreatment of UT7/TPO cells with a phosphatidylinositol 3-kinase (PI3K) inhibitor resulted in the cleavage of Bcl-xL even in the presence of TPO. We thus hypothesized that PI3K or its downstream signaling molecule inhibits the activation of caspase-3 and consequent cleavage of Bcl-xL. To prove this possibility, we prepared UT7/TPO cells transfected with constitutively active Akt-1. When TPO was depleted, the transfectant was significantly less liable to caspase-3 activation and Bcl-xL cleavage. Concerning transcriptional regulation of Bcl-xL, suppression of GATA-1 in UT7/TPO using siRNA caused decreased expression of both c-Mpl and Bcl-xL. Taken together, we conclude that GATA-1 regulates the expression of both c-Mpl and Bcl-xL, and once Bcl-xL is expressed, its protein level is maintained by the TPO-mediated Akt activation.