Continuous expression of Bcl-xL protein during megakaryopoiesis is post-translationally regulated by thrombopoietin-mediated Akt activation, which prevents the cleavage of Bcl-xL
Continuous expression of Bcl-xL protein during megakaryopoiesis is post-translationally regulated by thrombopoietin-mediated Akt activation, which prevents the cleavage of Bcl-xL
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DOI：
10.1111/j.1538-7836.2007.02546.x
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发表时间：
2007-06-01
影响因子：
10.4
通讯作者：
Nagasawa, T.
中科院分区：
文献类型：
--
作者：
Kozuma, Y.;Kojima, H.;Nagasawa, T.
背景：血小板生成素（TPO）的重要生物学活性之一是防止巨核细胞凋亡。由于抗凋亡蛋白Bcl-xL已被证明是红系分化所必需的，也在巨核细胞中大量表达，因此认为Bcl-xL在巨核细胞生成中起重要作用。目的：研究Bcl-xL在巨核细胞发生过程中的表达及其调控机制。方法和结果：在干细胞来源的巨核细胞中，Bcl-xL的表达在分化的早期和中期增加。无论是在体外细胞培养和在体内的动物模型中，Bcl-xL蛋白的表达一直维持到血小板生成阶段。TPO耗竭引起巨核细胞和TPO依赖的巨核细胞UT-7/TPO中Bcl-xL蛋白水平显著下降，而其mRNA水平无明显变化，表明TPO依赖的巨核细胞中Bcl-xL蛋白水平受到抑制后的调节。与这一发现一致，我们认识到TPO耗尽后出现12 kD的Bcl-xL片段。这种Bcl-xL的裂解被半胱天冬酶-3特异性抑制剂抑制。此外，用磷脂酰肌醇3-激酶（PI3 K）抑制剂预处理UT-7/TPO导致Bcl-xL的裂解，即使在TPO存在下。因此，我们假设PI3 K抑制半胱天冬酶-3的活化和随后的Bcl-xL裂解。为了证明这一点，我们准备了UT-7/TPO细胞转染组成型活性Akt-1。当TPO被耗尽时，转染子明显不容易被caspase-3激活和Bcl-xL切割。结论：Bcl-xL蛋白在整个巨核细胞生成过程中表达，直到产生血小板，并且其表达水平至少部分地通过TPO介导的Akt活化进行后调节。
Background: One of the important biological activities of thrombopoietin (TPO) is to prevent the apoptosis of megakaryocytes. As the antiapoptotic protein Bcl-xL, which has been proven to be indispensable for erythroid differentiation, is also abundantly expressed in megakaryocytes, it is assumed that Bcl-xL plays an important role in megakaryopoiesis. Objectives: We investigated the expression of Bcl-xL during megakaryopoiesis and the underlying regulatory mechanism. Methods and Results: In stem cell-derived megakaryocytes, expression of Bcl-xL increased in the early and mid-stages of the differentiation. Both in vitro in cell culture and in vivo in an animal model, expression of Bcl-xL protein was maintained until the platelet-producing stage. TPO depletion caused significant decrease in Bcl-xL protein level without affecting its mRNA in both megakaryocytes and TPO-dependent megakaryocytic UT-7/TPO cells, suggesting that Bcl-xL protein level in TPO-dependent cells is post-translationally regulated. In agreement with this finding, we recognized the appearance of a 12-kD fragment of Bcl-xL upon TPO depletion. This cleavage of Bcl-xL was inhibited by a caspase-3-specific inhibitor. Furthermore, pretreatment of UT-7/TPO with a phosphatidylinositol 3-kinase (PI3 K) inhibitor resulted in the cleavage of Bcl-xL even in the presence of TPO. We thus hypothesized that PI3 K inhibits the activation of caspase-3 and consequent cleavage of Bcl-xL. To prove this, we prepared UT-7/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. Conclusions: Bcl-xL protein is expressed throughout megakaryopoiesis until platelets are produced, and its expression level is at least in part post-translationally regulated through TPO-mediated Akt activation.