BH3-only Activator Proteins Bid and Bim Are Dispensable for Bak/Bax-dependent Thrombocyte Apoptosis Induced by Bcl-xL Deficiency

Abstract

A pivotal step in the mitochondrial pathway of apoptosis is activation of Bak and Bax, although the molecular mechanism remains controversial. To examine whether mitochondrial apoptosis can be induced by just a lack of antiapoptotic Bcl-2-like proteins or requires direct activators of the BH3-only proteins including Bid and Bim, we studied the molecular requisites for platelet apoptosis induced by Bcl-xL deficiency. Severe thrombocytopenia induced by thrombocyte-specific Bcl-xL knock-out was fully rescued in a Bak and Bax double knock-out background but not with single knock-out of either one. In sharp contrast, deficiency of either Bid, Bim, or both did not alleviate thrombocytopenia in Bcl-xL knock-out mice. An in vitro study revealed that ABT-737, a Bad mimetic, induced platelet apoptosis in association with a conformational change of the amino terminus, translocation from the cytosol to mitochondria, and homo-oligomerization of Bax. ABT-737-induced Bax activation and apoptosis were also observed in Bid/Bim-deficient platelets. Human platelets, upon storage, underwent spontaneous apoptosis with a gradual decline of Bcl-xL expression despite a decrease in Bid and Bim expression. Apoptosis was attenuated in Bak/Bax-deficient or Bcl-xL-overexpressing platelets but not in Bid/Bim-deficient platelets upon storage. In conclusion, platelet lifespan is regulated by a fine balance between anti-and proapoptotic multidomain Bcl-2 family proteins. Despite residing in platelets, BH3-only activator proteins Bid and Bim are dispensable for Bax activation and mitochondrial apoptosis.Platelets are unique blood cells that do not have a nucleus but contain mitochondria and have the daily job of handling hemostasis and thrombosis (1). They are produced from megakaryocytes and once released into circulation can function for about 10 days in humans and 4 -5 days in mice (2). They are then thought to be destroyed by the reticuloendothelial system. Regarding the mechanism that controls their lifespan, several studies have observed a decrease in mitochondrial membrane potential, caspase activation, and phosphatidylserine exposure in platelets, leading to the conclusion that platelets undergo apoptotic cell death (3-5). It has been demonstrated that platelets contain several apoptosis-related proteins such as Bcl-2 family proteins and a variety of caspase family proteins (3-7). Recently, Mason et al. (8) reported that knock-out of a single allele of the bcl-x gene results in mild thrombocytopenia, which is ameliorated in a Bak knock-out background. We have also reported previously that thrombocyte-specific homozygous Bcl-xL knock-out mice show marked thrombocytopenia (9). These findings established the critical role of Bcl-2 family proteins in regulating platelet apoptosis and lifespan. Platelets may be the simplest model for the study of Bcl-2 biology with physiological relevance because they neither perform de novo protein synthesis nor undergo proliferation.The proapoptotic multidomain Bcl-2 family proteins Bak and Bax serve as ef...

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

“…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. 11,12 As previously reported, despite severe thrombocytopenia, Bcl-xL knockout mice were born at the expected Mendelian frequency and did not show any gross abnormality; 15 analysis at embryonic day (ED) 13.5 also revealed that Bcl-xL knockout embryos grew up normally at the expected Mendelian frequency (Supplementary Table 1). We confirmed that Bcl-xL expression was efficiently diminished in the cultured megakaryocytes of the knockout mice ( Figure 1j) as well as in the platelets.…”

“…It should be noted that Bak deficiency caused modest thrombocytosis due to the prolonged lifespan of their platelets. 11,12 Mature megakaryocyte and platelet counts of the quadruple knockout mice were not significantly different from the control Bak knockout littermates (Figures 3b-d). The TUNEL-positive cell ratio of mature megakaryocytes in the quadruple knockout mice was very low and not different from their littermates (Figure 3e), suggesting that mature megakaryocyte apoptosis in the absence of both Bcl-xL and Mcl-1 could be prevented in a Bak and Bax knockout background.…”

“…Nevertheless, platelets possess Bcl-xL alone, unlike megakaryocytes in which two functional anti-apoptotic proteins, Bcl-xL and Mcl-1, are present. 11,12,16 We thus studied the effect of post-transcriptional degradation of Mcl-1 in platelets in vivo. At 1 or 2 days after the administration to wild-type mice of MG-132, a proteasome inhibitor, western blot revealed that the Mcl-1 protein was observed in platelets isolated from the MG-132-treated mice in contrast to platelets from the vehicle-treated mice (Figure 6a).…”

“…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. 12 Genetic studies deleting other anti-apoptotic Bcl-2 family genes have not reported any abnormality of the megakaryocytic lineage.…”

Deficiency in Bim, Bid and Bbc3 (Puma) do not prevent axonal injury induced death

“…Interestingly, lowering levels of pro-survival Bcl-2 family members alone can lead to apoptosis. 8 Necroptotic cell death is also dependent on BAX 9 and observed in injured neurons, but may not require BIM, BBC3, and BID. Finally, it is possible that BAX activation could be occurring independently of the Bcl-2 family (e.g.…”