Abstract
Mitochondrial apoptosis regulates survival and development of hematopoietic cells. Prominent roles of some Bcl-2-family members in this regulation have been established, for instance for pro-apoptotic Bim and anti-apoptotic Mcl-1. Additional, mostly smaller roles are known for other Bcl-2-members but it has been extremely difficult to obtain a comprehensive picture of the regulation of mitochondrial apoptosis in hematopoietic cells by Bcl-2-family proteins. We here use a system of mouse ‘conditionally immortalized’ lymphoid-primed hematopoietic progenitor (LMPP) cells that can be differentiated in vitro to pro-B cells, to analyze the importance of these proteins in cell survival. We established cells deficient in Bim, Noxa, Bim/Noxa, Bim/Puma, Bim/Bmf, Bax, Bak or Bax/Bak and use specific inhibitors of Bcl-2, Bcl-XL and Mcl-1 to assess their importance. In progenitor (LMPP) cells, we found an important role of Noxa, alone and together with Bim. Cell death induced by inhibition of Bcl-2 and Bcl-XL entirely depended on Bim and could be implemented by Bax and by Bak. Inhibition of Mcl-1 caused apoptosis that was independent of Bim but strongly depended on Noxa and was completely prevented by the absence of Bax; small amounts of anti-apoptotic proteins were co-immunoprecipitated with Bim. During differentiation to pro-B cells, substantial changes in the expression of Bcl-2-family proteins were seen, and Bcl-2, Bcl-XL and Mcl-1 were all partially in complexes with Bim. In differentiated cells, Noxa appeared to have lost all importance while the loss of Bim and Puma provided protection. The results strongly suggest that the main role of Bim in these hematopoietic cells is the neutralization of Mcl-1, identify a number of likely molecular events during the maintenance of survival and the induction of apoptosis in mouse hematopoietic progenitor cells, and provide data on the regulation of expression and importance of these proteins during differentiation along the B cell lineage.
Highlights
Mitochondrial apoptosis regulates many biological processes and is very important for differentiation and regulation of survival in hematopoietic cells [1]
The results strongly suggest that the main role of Bim in these hematopoietic cells is the neutralization of Mcl-1, identify a number of likely molecular events during the maintenance of survival and the induction of apoptosis in mouse hematopoietic progenitor cells, and provide data on the regulation of expression and importance of these proteins during differentiation along the B cell lineage
Mitochondrial apoptosis is regulated by the Bcl-2-family, comprising three groups of proteins that can be distinguished by their structure and by their function
Summary
Mitochondrial apoptosis regulates many biological processes and is very important for differentiation and regulation of survival in hematopoietic cells [1]. Mitochondrial apoptosis is regulated by the Bcl-2-family, comprising three groups of proteins that can be distinguished by their structure and by their function. The proapoptotic group of Bax and Bak serve as the effectors, initiating the activation of effector caspases in the cytosol. The five antiapoptotic proteins, Bcl-2, Bcl-XL, Bcl-w, Mcl-1 and A1, inhibit apoptosis by binding pro-apoptotic family proteins. The third group, known as BH3-only proteins and made up by eight members, are the initiators of apoptosis. BH3-only proteins trigger apoptosis through one of two mechanisms, by inhibiting antiapoptotic Bcl-2-proteins and/or directly activating Bax/Bak. At least three BH3-only proteins, Bim, tBid (the active form of Bid) and Puma, can directly activate, while all BH3-only proteins (including Noxa, Bad, Bmf, Bik and Hrk) can inhibit anti-apoptotic Bcl-2-proteins [1, 2]
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