Continuous expression of Bcl‐xL protein during megakaryopoiesis is post‐translationally regulated by thrombopoietin‐mediated Akt activation, which prevents the cleavage of Bcl‐xL

Abstract

Summary. 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.

Mcl-1 and Bcl-xL regulate Bak/Bax-dependent apoptosis of the megakaryocytic lineage at multistages

“…With regard to Bcl-xL, previous in-vitro studies reported not only its expression profile during megakaryocyte differentiation but also its anti-apoptotic involvement. 10,31 This agrees with our in-vitro study showing moderate apoptosis of megakaryoblastic cells upon ABT-737 treatment (Figures 5b-e). On the other hand, in in-vivo settings, Josefsson et al 16 very recently analyzed this in detail and reported that Bcl-xL played an important role in preventing mature megakaryocytes from apoptosis at the proplatelet formation stage but was not required for their growth and development.…”

“…We next investigated the involvement of Bcl-xL, another anti-apoptotic Bcl-2 family protein known to exist in megakaryocytes, 10 in the development and survival of the megakaryocytic lineage. We used previously generated megakaryocytic lineage-specific homozygote Bcl-xL knockout mice (bcl-x flox/flox pf4-cre), 15 which presented with severe thrombocytopenia due to massive platelet apoptosis.…”

“…3 However, the relationship of Mcl-1 with the terminally differentiated megakaryocytes has not been well understood, with the exception of a report describing the existence of Mcl-1 protein in megakaryocytes. 9 Bcl-xL is also continuously expressed in the megakaryocytic lineage during megakaryopoiesis 10 and is required for platelet survival. 11 However, mature megakaryocytes increased in mice with genetic deletion of Bcl-xL.…”

Caspase activation is involved in early megakaryocyte differentiation but not in platelet production from megakaryocytes

“…8,9 Furthermore, bcl-2-or bcl-xL-transfected MKs, which should be resistant to caspase-3 activation, were reported to be significantly less capable of forming proplatelets. 9,10 Contrary to these findings, our recent in vitro and in vivo observations suggested that Bcl-xL protein is highly expressed throughout megakaryopoiesis until the platelet-producing late stage of maturation, 11 which raised the question about the apoptosis theory of platelet production. On the other hand, recent studies have established that caspase activation is involved in nonapoptotic cellular functions, such as the proliferation and differentiation of various types of cells.…”

Megakaryocytes possess a functional intrinsic apoptosis pathway that must be restrained to survive and produce platelets

“…If megakaryocytes are susceptible to activation of the intrinsic pathway to apoptosis, then one would expect that one or more members of the Bcl-2 family of prosurvival proteins must keep Bak and Bax in check to maintain cellular integrity. Given the known role of one such prosurvival, Bcl-x L , in regulating platelet life span (Mason et al, 2007), and given its documented expression in megakaryocytes (Terui et al, 1998;Sanz et al, 2001;Kozuma et al, 2007), we generated a megakaryocyte-specific deletion of Bcl-x. Mice harboring a (Fig.…”