Loss of Bak enhances lymphocytosis but does not ameliorate thrombocytopaenia in BCL-2 transgenic mice

Bax and Bak are critical effectors of apoptosis. Although both are widely expressed and usually functionally redundant, recent studies suggest that Bak has particular importance in certain

cell types. Genetic and biochemical studies indicate that Bak activation is prevented primarily by Mcl-1 and Bcl-xL, whereas Bax is held in check by all pro-survival Bcl-2 homologues,

including Bcl-2 itself. In this study, we have investigated whether loss of Bak or elevated Mcl-1 modulates haemopoietic abnormalities provoked by overexpression of Bcl-2. The Mcl-1

transgene had little impact, probably because the expression level was insufficient to effectively reduce Bak activation. However, loss of Bak enhanced lymphocytosis in vavP-BCL-2 transgenic

mice and increased resistance of their thymocytes to some cytotoxic agents, implying that Bak-specific signals can be triggered in certain lymphoid populations. Nevertheless, lack of Bak

had no significant impact on thymic abnormalities in vavP-BCL-2tg mice, which kinetic analysis suggested was due to accumulation of self-reactive thymocytes that resist deletion.

Intriguingly, although Bak−/− mice have elevated platelet counts, Bak−/−vavP-BCL-2 mice, like vavP-BCL-2 littermates, were thrombocytopaenic. To clarify why, the vavP-BCL-2 platelet

phenotype was scrutinised more closely. Platelet life span was found to be elevated in vavP-BCL-2 mice, which should have provoked thrombocytosis, as in Bak−/− mice. Analysis of bone marrow

chimaeric mice suggested the low platelet phenotype was due principally to extrinsic factors. Following splenectomy, blood platelets remained lower in vavP-BCL-2 than wild-type mice.

However, in Rag1−/− BCL-2tg mice, platelet levels were normal, implying that elevated lymphocytes are primarily responsible for BCL-2tg-induced thrombocytopaenia.

Apoptosis has a vital role in regulating cell numbers during haemopoiesis and failure to remove damaged, superfluous or potentially dangerous cells can lead to malignancy or autoimmunity.

Many critical life-or-death checkpoints, particularly during lymphopoiesis, are governed by opposing factions of the Bcl-2 protein family, which regulate the ‘intrinsic’ apoptosis pathway.1,

2 Bcl-2, discovered via the t(14;18) chromosome translocation typical of human follicular lymphoma,3, 4, 5 inhibits apoptosis,6 as do its closest homologues (Bcl-xL, Bcl-w, A1/Bfl1, Mcl-1

and, in humans, Bcl-B). Other close homologues (Bax and Bak) instead promote apoptosis, as do distant relatives known as BH3 (Bcl-2 homology region 3)-only proteins because they share only

an ∼26 amino-acid motif with the wider Bcl-2 family. During cellular stress, BH3-only proteins are induced and bind with high avidity via their amphipathic BH3 α-helix to the hydrophobic

groove on the surface of pro-survival Bcl-2-like proteins,7, 8, 9 thereby preventing them from restraining any activated Bax or Bak molecules. Certain BH3-only proteins (particularly Bim and

cleaved Bid) can also bind weakly and transiently to Bax and/or Bak, triggering their conformational change and subsequent homo-oligomerisation on the outer mitochondrial membrane. As a

consequence, cytochrome c is released into the cytoplasm, leading to the activation of the proteases (caspases) that provoke cellular demolition by cleaving vital proteins.

Although Bax and Bak are both widely expressed and functionally redundant,10 recent studies suggest Bak may have particular importance in certain cell types. Thus, loss of Bak results in

thrombocytosis, whereas loss of Bax does not, indicating that Bak has the more important role in regulating platelet life span.11, 12 Furthermore, loss of Bak was able to partially rescue

thymic defects caused by conditional deletion of Mcl-1, whereas neither overexpression of Bcl-2 nor loss of Bax was able to do so.13 Specificity of interactions may account for these

observations: Bak binds tightly to Mcl-1 and Bcl-xL but only poorly to Bcl-2, whereas Bax binds avidly to all the pro-survival proteins.14, 15, 16 Presumably, therefore, Bax activation can

be thwarted by all pro-survival proteins, including Bcl-2, whereas Bak is kept in check by Mcl-1 and Bcl-xL.

To further explore the role of Bak-specific cell death during haemopoiesis, we have investigated whether loss of Bak or increased expression of Mcl-1 enhances the impact of pan-haemopoietic

overexpression of Bcl-2. To do so, we crossed vavP-BCL-2 transgenic (hereafter BCL-2tg) mice17 with Bak−/−10 or vavP-Mcl-1 transgenic (hereafter Mcl-1tg) mice18 and compared the phenotypes

of single and doubly mutant offspring. This study also enabled us to undertake further analysis of the puzzling thymic and platelet phenotype of BCL-2tg mice.

Overexpression of Bcl-2 via the haemopoietic cell-specific vavP-driven transgene19 enhances the survival of T- and B-lymphoid cells, which accumulate in excessive numbers in the

periphery.17, 20 In addition, BCL-2tg mice have a distinctive thymic phenotype: a reduced proportion of pre-T cells (CD4+CD8+ double positive; hereafter DP) and elevated proportions of the

other three major populations (CD4−CD8− double negative, hereafter DN; CD4+CD8− single positive, hereafter CD4SP; and CD4−CD8+ single positive, hereafter CD8SP).17 This is referred to as the

‘low DP’ thymic phenotype of BCL-2tg mice (see further below).

To assess whether overexpression of Mcl-1 exacerbates the BCL-2tg phenotype, we compared the composition of blood and haemopoietic tissues in 6-week-old neonates. Despite a minor (P≤0.05)

increase in the total number of DP thymocytes in Mcl-1/BCL-2 bi-transgenic compared with BCL-2tg mice, the proportion of DP thymocytes was comparably low (lower left panel in Supplementary

Figure 1B) and there was little impact on the BCL-2tg-induced lymphocytosis in the other lymphoid organs (Supplementary Figure 1 and Supplementary Table 1).

To assess the impact of loss of Bak, we first analysed haemopoietic tissues in young adult (12- to 14-week-old) mice. Loss of Bak had little impact alone, but did increase lymphocytosis in

BCL-2tg mice (Figure 1 and Supplementary Table 2). This was most apparent in the spleen where cellularity, already elevated approximately fivefold in BCL-2tg mice, increased to nearly

sevenfold in Bak−/− BCL-2tg animals, due primarily to a further elevation in the number of immunoglobulin (Ig) isotype-switched (B220+IgM−IgD−) B-lymphoid cells and mature T cells (CD4+ and

CD8+; Figure 1a). A comparable cross of Bax−/− and BCL-2tg mice revealed no comparable differences between BCL-2tg and Bax−/− BCL-2tg animals (Supplementary Table 3).

Loss of Bak exacerbates lymphocytosis in BCL-2tg mice. Enumeration of total leukocytes and indicated lymphoid populations in the (a) spleen and (b) thymus of 12- to 14-week-old male mice

(n=6–10 per genotype: WT, white; Bak−/−, light grey; BCL-2tg, dark grey; Bak−/−BCL-2tg, black). B220+IgM/D+ indicates B220+ cells that are IgM+ and/or IgD+. Bars represent mean±S.E.M.; see

also Supplementary Table 2. Statistical significance is shown only for BCL-2tg versus Bak−/−BCL-2tg; *P