ALBERT

Description: Megakaryocytes (Mgk) differentiate from hematopoietic stem cells through the complex process of endomitosis and cytoplasmic development, and finally form proplatelets in the terminal stage of the differentiation. To elucidate the role of a proapoptotic effecter protein, caspase-3, in megakaryopoiesis and thrombopiesis, we analyzed the phenotype of Mgk in vav-Bcl-2 transgenic (Tg) mice, which overexpress Bcl-2 exclusively in hematopoietic cells by the regulation of vav promotor. As was expected, Bcl-2-overexressing Mgk were remarkably resistant to caspase-3 activation induced by the depletion of thrombopoietin (TPO) from the culture. Percentages of proplatelet formation (PPF) assayed by culturing primary Mgk were slightly increased in the Tg mice. Platelet production from cultured Mgk, which was assayed by flowcytometry, was also slightly increased, indicating normal ability for Plt production from Bcl-2-overexpressing mature Mgk. To further analyze the ability for thrombopoiesis, we assayed the trends of Plt recovery following experimental thrombocytopenia. As Bcl-2-overexpressing mice had giant splenomegaly, we used splenectomized mice, which showed normal Plt counts. After the injection of 5-FU, we observed significant delay of the Plt recovery in the Tg mice, as well as the lack of the overshoot of Plt counts during the recovery phase. To gain insights about the mechanism underlying the delayed Plt recovery, we analyzeded the ploidy of DNA content. In the steady state, peak ploidy of Mgk obtained from the bone marrow of the Tg mice shifted to 2–4N. In a sharp contrast, in Plt recovery phase after 5-FU injection, peak ploidy shifted to 16–32N, and the percentages of Mgk possessing less than 16N ploidy were dramatically decreased. Consistent with this finding, we observed the delay of PPF in Bcl-2-overexpressing cells when Mgk were derived from c-kit+/lineage- progenitor cells in the presence of TPO. These data indicate that differentiation of immature Mgk into mature Plt-producing stage is significantly delayed in the Tg mice, but once the maturation proceeds, Mgk normally produce Plt. Taken together, we conclude that caspase-3 activation is involved in the early stage of the differentiation of Mgk, but not in Plt production.

Description: 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.