Our studies demonstrate that megakaryocytes possess an intrinsic apoptosis pathway, the key components of which are prosurvival Bcl-xL, and prodeath Bak and Bax. Contrary to the widely accepted model, this pathway is not activated by megakaryocytes to facilitate platelet shedding. Deletion of Bak and Bax, the gatekeepers of the intrinsic apoptosis pathway, had no adverse effect on megakaryocyte number, ploidy, or proplatelet formation. Platelet production at steady state, or in response to experimentally induced thrombocytopenia, was normal. This was true whether Bak and Bax were conditionally or constitutively deleted from the megakaryocyte lineage. The only abnormality we observed was an increase in the number of naked megakaryocyte nuclei when both Bak and Bax were deleted. Although apparent at steady state, the phenomenon was much more pronounced during recovery from thrombocytopenia. This suggests that Bak and Bax mediate the clearance of the megakaryocyte nucleus after platelet shedding. Whether or not this is an apoptotic process is unclear. It is certainly possible; we observed a thin ring of cytoplasm around naked nuclei, perhaps this is normally the site of postshedding apoptosis designed to ensure swift engulfment. In support of this notion, our live cell imaging data demonstrated that, in vitro, wild-type megakaryocytes expose significant levels of PS once proplatelet formation is complete.
In stark contrast to their absence, the activation of Bak and Bax had a profound and detrimental impact on megakaryocytes. Wild-type cells treated in vitro with the proapoptotic agent ABT-737, which inhibits Bcl-xL
, Bcl-2, and Bcl-w (Oltersdorf et al., 2005
), suffered mitochondrial damage, caspase activation, and death. A similar phenomenon was observed in mature, unmanipulated megakaryocytes lacking Bcl-xL
. In both cases, proplatelet formation failed. Genetic deletion of Bak and Bax could block death and rescue proplatelet formation in the presence of ABT-737, and completely restored platelet production in mice lacking Bcl-xL
. Thus, megakaryocytes require Bcl-xL
to restrain the activity of Bak and Bax during platelet production.
Our data indicate that megakaryocytes become dependent on Bcl-xL just as they enter into proplatelet formation. It is not required for their growth and development, as indicated by the abundance of polyploid megakaryocytes in the BM of Bcl-xPf4Δ/Pf4Δ mice, and in the yield of large mature cells from cultures of Bcl-xL–deficient BM and fetal liver. Unlike their wild-type counterparts, however, cultured Bcl-xPf4Δ/Pf4Δ megakaryocytes are unable to elaborate proplatelets, instead undergoing Bak/Bax-dependent death which is accompanied by significant exposure of PS. Although the situation in vivo is more difficult to visualize, there is no doubt that platelet production is disturbed, independently of the severe thrombocytopenia that results from loss of Bcl-xL in platelets. This is demonstrated by the ultrastructural analysis, and also the BM chimera experiments which confirmed that even in the presence of wild-type hematopoiesis, Bcl-xPf4Δ/Pf4Δ megakaryocytes produce morphologically aberrant platelets.
Clearly, induction of Bak- and Bax-mediated apoptosis results in a failure of platelet shedding, but it appears there are additional mechanisms by which proapoptotic stimuli can block proplatelet formation. Although Bak- and Bax-deficient megakaryocytes were resistant to etoposide-induced mitochondrial damage and caspase activation, they still exhibited a failure of proplatelet formation. This suggests that DNA damage may suppress platelet shedding independently of apoptosis occurring. Etoposide treatment results in DNA double-strand and single-strand breaks (Wozniak and Ross, 1983
), which trigger activation of kinase signaling cascades, primarily the ATM–Chk2 and ATR–Chk1 pathways (Cimprich and Cortez, 2008
). The role of these pathways in megakaryocytes, and the mechanism by which they might influence proplatelet formation is yet to be established. Interestingly, even in the absence of Bak and Bax, STS, a classical inducer of the intrinsic apoptosis pathway, was able to induce mitochondrial damage and caspase activation in megakaryocytes. This contrasts with Bak−/− Bax−/−
mouse embryonic fibroblasts, which are resistant (Wei et al., 2001
). These data indicate that megakaryocytes possess additional cell death signaling pathways, and, as Q-VD-OPh could not block STS-induced mitochondrial damage, they are likely to be caspase independent.
Our findings contradict a substantial body of literature which holds that megakaryocytes deliberately use the apoptotic machinery to facilitate platelet production. We believe that many of the apparent discrepancies can be explained. First, several of the key studies reporting that impaired apoptotic signaling diminishes platelet production involved overexpression of Bcl-2 family survival proteins, either in mice or in cell culture (Ogilvy et al., 1999
; De Botton et al., 2002
; Kaluzhny et al., 2002
). Overexpression of any protein can impact on cellular processes in complex and unforeseen ways, and Bcl-2 has been linked to the cell cycle (Vairo et al., 1996
), calcium homeostasis (Bassik et al., 2004
), autophagy (Pattingre et al., 2005
), and inflammasome function (Bruey et al., 2007
). Indeed, it has recently been demonstrated that Bcl-2 transgenic mice, which exhibit thrombocytopenia (Ogilvy et al., 1999
), recover normal platelet counts upon splenectomy (Kozuma et al., 2009
). Second, data from in vitro culture systems, cell lines, and chemical inhibitor studies should be interpreted with caution. Our results agree with previous studies that high concentrations of the caspase inhibitor zVAD.fmk can inhibit proplatelet formation by megakaryocytes (De Botton et al., 2002
; Clarke et al., 2003
). In contrast, however, we found that the difluorophenoxy-methylketone-based pan-caspase inhibitor Q-VD-OPh does not. Wild-type proplatelets in cultures treated with Q-VD-OPh survived significantly longer than untreated counterparts. Given that proplatelets lacking Bak and Bax exhibited a similar increase in stability and survival, we believe the most likely explanation for the discrepancy between the two inhibitors is that high doses of z-VAD.fmk trigger caspase-independent toxicity in megakaryocytes. It is known that z-VAD.fmk has potent activity against cathepsins B, H, and L (Chauvier et al., 2007
), and in some cells can lead to necrosis (Temkin et al., 2006
; Wu et al., 2011
). In fact, in 2009 the Nomenclature Committee on Cell Death recommended that the term z-VAD.fmk-inhibitable should be used in preference to “caspase-dependent” (Kroemer et al., 2009
In addition to the intrinsic apoptosis pathway, the extrinsic pathway has also been implicated in platelet shedding. Treatment of MEG-01 cells with Fas ligand (FasL) or an anti-Fas agonistic antibody was reported to increase proplatelet formation and production of platelet-like particles (Clarke et al., 2003
). Similar results were obtained with primary mouse megakaryocytes or human bone core explants subjected to Fas agonism. This might suggest that although the intrinsic pathway is dispensable for platelet shedding, either the extrinsic pathway alone is required, or a combination of both is essential. Our results with Q-VD-OPh argue against this notion, as Fas signaling is mediated by caspase-8 (Boldin et al., 1996
; Fernandes-Alnemri et al., 1996
; Muzio et al., 1996
) and Q-VD-OPh is an effective inhibitor of this critical initiator (Chauvier et al., 2007
). Genetic deletion studies will be required to define the role of the extrinsic apoptosis pathway in megakaryocyte biology.
Our results with carboplatin demonstrate that deletion of Bak and Bax can significantly protect the megakaryocyte lineage against a pathophysiological insult in vivo.
Chemotherapy-induced thrombocytopenia (CIT) remains a significant unmet clinical need (Vadhan-Raj, 2009
), and it will be interesting to see whether Bak/Bax-mediated killing contributes to CIT caused by other agents. In addition to cytotoxic drugs, it is conceivable the pathway might also be activated by autoantibodies, viral infections such as HIV, or radiation. In support of the latter notion, ablation of Bak and Bax in hematopoietic and endothelial cells was recently shown to protect the BM and increase the survival of mice exposed to 12.5 Gy of whole-body irradiation (Kirsch et al., 2010
). Whether loss of Bak and Bax can specifically protect the megakaryocyte lineage against radiation remains to be established.
Because carboplatin causes the death not just of megakaryocytes, but of megakaryoblasts and megakaryocyte progenitors (Zeuner et al., 2007
), the protection conferred by loss of Bak and Bax suggests that the intrinsic pathway must be restrained throughout the developing megakaryocyte lineage. Prosurvival proteins expressed in megakaryocytes include Bcl-xL
, Bcl-2, and Mcl-1 (Sanz et al., 2001
; Zeuner et al., 2007
; this study). One explanation for the fact that loss of Bcl-xL
only appears to affect megakaryocytes undergoing platelet shedding might be that it is the sole prosurvival protein expressed at this critical juncture. The heterogeneous nature of megakaryocyte cultures makes this a difficult notion to test, and the fact that platelets express Bcl-2 suggests otherwise. Perhaps a more likely scenario is that polyploidization, proplatelet formation, and platelet shedding generate acute proapoptotic stresses within the megakaryocyte, which multiple Bcl-2 family prosurvival proteins are required to resist. Loss of Bcl-xL
is enough to disturb the pro- and antiapoptotic balance. It will be interesting to see whether deletion of Mcl-1 and Bcl-2 in megakaryocytes results in a similar phenotype. Given that agents specifically targeting Bcl-2 family prosurvival proteins are currently being developed for use in a range of human malignancies (Leber et al., 2010
; Petros et al., 2010
; Wilson et al., 2010
; Roberts et al., In Press
), it will be important to understand the contribution they make to the development and survival of the megakaryocyte lineage.