Mcl-1 and Bcl-xL coordinately regulate megakaryocyte survival

Abstract

Mature megakaryocytes depend on the function of Bcl-x L , a member of the Bcl-2 family of prosurvival proteins, to proceed safely through the process of platelet shedding. Despite this, loss of Bcl-x L does not prevent the growth and maturation of megakaryocytes, suggesting redundancy with other prosurvival proteins. We therefore generated mice with a megakaryocyte-specific deletion of Mcl-1, which is known to be expressed in megakaryocytes. Megakaryopoiesis, platelet production, and platelet lifespan were unperturbed in Mcl IntroductionMegakaryocytes are large polyploid cells responsible for the production of blood platelets. They develop primarily in the BM and spleen. On reaching maturity, megakaryocytes extend long pseudopodial projections called proplatelets into the circulation, and it is from these structures that platelets are released. [1][2][3] Once born, platelets have a brief lifespan in the circulation: 10 days in humans, 5 days in mice. 4,5 In recent years, it has become apparent that this finite existence is governed by the interplay between members of the Bcl-2 protein family, the critical regulators of the "intrinsic" or "mitochondrial" apoptosis pathway. 6 Platelets depend on the prosurvival protein Bcl-x L to maintain their viability. 7 Mutations in murine Bcl-x L cause dose-dependent cell-intrinsic reductions in platelet lifespan. 7-9 Pharmacologic blockade of Bcl-x L with the BH3 mimetic drugs ABT-737 10 or ABT-263 11 triggers platelet death 7,12-15 and thrombocytopenia in mice, 7,16 dogs, 15 and humans. 17,18 The function of Bcl-x L is to restrain the prodeath proteins Bak and Bax. When activated, Bak and Bax induce mitochondrial outer membrane permeabilization (MOMP), resulting in platelet apoptosis, which, at least in vitro, is characterized by cytochrome c release, caspase activation, and phosphatidylserine exposure. [12][13][14][15] At steady state in vivo, aged platelets that have escaped hemostatic consumption undergo Bak-mediated apoptosis and clearance from the circulation. Genetic deletion of Bak and Bax almost doubles platelet lifespan, 9 rescues the thrombocytopenia caused by loss of Bcl-x L , 9 and renders platelets refractory to the effects of ABT-737. 13 We recently demonstrated that Bcl-x L is also essential for mature megakaryocytes to proceed safely through the process of platelet shedding. 9 In vitro, loss of Bcl-x L triggers Bak-and Bax-mediated mitochondrial damage, caspase activation, and failure of proplatelet formation. In vivo, mature, shedding megakaryocytes lacking Bcl-x L exhibit severe dysmorphology and produce grossly abnormal platelets that survive only hours in the circulation. Bcl-x L is, however, dispensable for the growth and development of megakaryocytes, suggesting that other Bcl-2 family prosurvival proteins are necessary to promote survival during this process. Of the 5 prosurvival members of the Bcl-2 family, 6 3 are known to be expressed in megakaryocytes: Bcl-2, Bcl-x L , and Mcl-1. 9 Given its critical role in hematopoietic progenitors and lymphocy...

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

“…These results indicated that, among the five members of the anti-apoptotic Bcl-2 family, either existence of Mcl-1 or Bcl-xL is required for the development and survival of megakaryocytes in both developing and adult mice. During revision of this manuscript, Debrincat et al 33 published a paper on line reporting the importance of these anti-apoptotic proteins in megakaryocyte survival, which agree with our current findings. Our present study now provides solid evidence that Mcl-1 and Bcl-xL have an important pro-survival role in preventing Bak/Bax-dependent megakaryocyte apoptosis in both developing and adult mice.…”

Platelet production proceeds independently of the intrinsic and extrinsic apoptosis pathways

“…However, staurosporine does not stimulate thrombopoiesis, rather, it causes mitochondrial damage, failure of proplatelet formation and death. Taken together, our results and those of several recent studies 18,22,[28][29][30]56,57 leave little room for the notion that apoptosis, as it is currently defined 54 , is required for, or can augment, platelet production by megakaryocytes. It remains possible that one or more components of the apoptotic machinery may play a role in the process, but convincing evidence for this has yet to emerge.…”

“…In megakaryocytes, MCL-1 and BCL-X L are both required to keep BAK and BAX in check 18,28,29 . Their simultaneous deletion leads to a failure of megakaryopoiesis, haemorrhage and embryonic lethality.…”

Getting out and about: the emergence and morphogenesis of the vertebrate lymphatic vasculature

“…CLEC-2 (CLEC1B -Mouse Genome Informatics), a C-type (9) lectin receptor on platelets that binds to podoplanin on LECs, is important for platelet aggregation at the junctions between developing lymph sacs and the CVs (Bertozzi et al, 2010;SuzukiInoue et al, 2010;Osada et al, 2012). Mice deficient in megakaryocytes, platelets or platelet aggregation, or with mutations disrupting podoplanin, O-glycosylation (a key post-translational modification of podoplanin), CLEC-2, or SLP-76 (LCP2-Mouse Genome Informatics) signalling in platelets, all exhibit blood-filled lymphatic vessels (Fu et al, 2008;Uhrin et al, 2010;Carromolino et al, 2010;Bertozzi et al, 2010;Suzuki-Inoue et al, 2010;Debrincat et al, 2012;Osada et al, 2012), reflecting the aberrant maintenance of blood-lymphatic vascular connections. Whether separation of the two vascular compartments is mediated solely by platelets acting as a physical barrier to 'plug' openings between veins and lymph sacs/lymphatic vessels, or whether platelet-LEC interaction results in downstream signalling events important for blood-lymphatic vascular separation remains to be established.…”