Combined loss of proapoptotic genes Bak or Bax with Bim synergizes to cause defects in hematopoiesis and in thymocyte apoptosis

Abstract

The proapoptotic members of the Bcl-2 family can be subdivided into members that contain several Bcl-2 homology (BH) domains and those that contain only the BH3 domain. Although it is known that BH3-only proteins and the multi-BH domain proteins, Bak and Bax, are essential for programmed cell death, the overlapping role of these two subgroups has not been examined in vivo. To investigate this, we generated Bak/Bim and Bax/Bim double deficient mice. We found that although Bax-/-Bim-/-, but not Bak-/-Bim-/-, mice display webbed hind and front paws and malocclusion of the incisors, both groups of mice present with dysregulated hematopoiesis. Combined loss of Bak and Bim or Bax and Bim causes defects in myeloid and B-lymphoid development that are more severe than those found in the single knock-out mice. Bak-/-Bim-/- mice have a complement of thymocytes that resembles those in control mice, whereas Bax-/-Bim-/- mice are more similar to Bim-/- mice. However, thymocytes isolated from Bak-/-Bim-/- or Bax-/-Bim-/- mice are markedly more resistant to apoptotic stimuli mediated by the intrinsic pathway as compared with thymocytes from single-knockout mice. These data suggest an essential overlapping role for Bak or Bax and Bim in the intrinsic apoptotic pathway.

Figure 1.

Bax/Bim DKO mice display developmental defects. (A) Photograph showing webbed feet in a Bax/Bim DKO (C57BL/6 background) mouse which is not seen in a WT mouse. (B) Photograph of Bax/Bim DKO runted mouse (white on a mixed C57BL/6/129sv background) compared with age- and sex-matched Bax^+/+Bim^−/− control (black) mouse. (C) Photograph of Bax/Bim DKO mouse (white on a mixed C57BL/6/129sv background) showing deformed incisors.

Figure 2.

Bak/Bim DKO and Bax/Bim DKO have normal numbers of tissue macrophages (A, B) Bak/Bim DKO and Bax/Bim DKO mice contain similar numbers of macrophages in their liver. Representative photomicrographs of F4/80-positive cells in the liver in WT (n = 13), Bak^−/− (n = 12), Bax^−/− (n = 7), Bim^−/− (n = 16), Bak/Bim DKO (n = 3), and Bax/Bim DKO (n = 7) mice. Values represent the mean ± standard error. (C) Quantitative analysis of F4/80-positive macrophages in the peritoneum. Peritoneal cells from WT (n = 23), Bak^−/− (n = 12), Bax^−/− (n = 9), Bim^−/− (n =15), Bak/Bim DKO (n = 4), and Bax/Bim DKO (n = 8) mice were analyzed for F4/80 antigen expression. Values represent the mean ± standard error, which were compared by Student's t test.

Figure 3.

Bax/Bim DKO mice display an altered CD4⁺8⁻/CD4⁻8⁺ T cell ratio resulting from abundant accumulation of mature CD4⁻8⁺ T cells. (A) Bak/Bim DKO and Bax/Bim DKO mice display a 1:1 CD4⁺8⁻/CD4⁻8⁺ T cell ratio in their spleen. Single-cell splenocyte suspensions were stained with the indicated antibodies and then analyzed by flow cytometry. Shown is a dot plot gated on the CD45⁺CD3⁺ splenocytes. (B) Bax/Bim DKO and Bim^−/− mice exhibit reduced percentages and numbers of CD4⁺8⁺ double positive thymocytes. Single-cell thymocyte suspensions were stained with the indicated antibodies and analyzed by flow cytometry.

Figure 4.

Bak/Bim DKO and Bax/Bim DKO thymocytes have abnormally low sensitivity to intrinsic apoptotic stimuli. Thymocytes isolated from WT, Bak^−/−, Bax^−/−, Bim^−/−, Bak/Bim DKO, and Bax/Bim DKO mice were plated in triplicate and left untreated (death by cytokine deprivation) or were treated with etoposide (7.5 ng/ml) or FasL (20 ng/ml) and analyzed by flow cytometry. All data are expressed as percentages of (A) Rh123-positive, (B) annexin V-negative, or (C) 7AAD-negative cells normalized to the 0 time point. Bak/Bim DKO and Bax/Bim DKO thymocytes display reduced activation of caspase-9 (D) and (E) caspase-3. Thymocytes isolated from WT, Bak^−/−, Bax^−/−, Bim^−/−, Bak/Bim DKO, and Bax/Bim DKO mice were left untreated (cytokine deprivation) or were treated for 8 h with etoposide and then analyzed for the activity of caspase-9 and -3. Values represent the mean ± standard error, which were compared by Student's t test. *P < 0.05 as compared with WT. All data are representative of two independently performed experiments. ^†P < 0.05 as compared with Bim^−/−.

Figure 5.

Summary of the genetic interactions of Bcl-2 family members. Previous studies have shown that deficiency in Bim, but not Bax, is sufficient to rescue Bcl-2^−/− mice. These data suggest that, in part, Bcl-2 may function to inhibit Bim. Bcl-2 also may function to prevent the formation of higher-order complexes of Bak and Bax or function to prevent the activation of Bak or Bax. Because Bim^−/− or Bcl-2/Bim DKO thymocytes undergo apoptosis in response to certain intrinsic apoptotic stimuli (e.g., DNA damage), these data suggest that additional pathways that are independent of Bim must exist. However, all of these intrinsic pathways require Bak and Bax for apoptosis.