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. 2013 Oct 18;288(42):30009-30018.
doi: 10.1074/jbc.M112.443093. Epub 2013 Aug 28.

The Bcl-2 homology domain 3 (BH3)-only proteins Bim and bid are functionally active and restrained by anti-apoptotic Bcl-2 family proteins in healthy liver

Takahiro Kodama  1 , Hayato Hikita  1 , Tsukasa Kawaguchi  1 , Yoshinobu Saito  1 , Satoshi Tanaka  1 , Minoru Shigekawa  1 , Satoshi Shimizu  1 , Wei Li  1 , Takuya Miyagi  1 , Tatsuya Kanto  1 , Naoki Hiramatsu  1 , Tomohide Tatsumi  1 , Tetsuo Takehara  2
Affiliations

The Bcl-2 homology domain 3 (BH3)-only proteins Bim and bid are functionally active and restrained by anti-apoptotic Bcl-2 family proteins in healthy liver

Takahiro Kodama et al. J Biol Chem. .

Abstract

An intrinsic pathway of apoptosis is regulated by the B-cell lymphoma-2 (Bcl-2) family proteins. We previously reported that a fine rheostatic balance between the anti- and pro-apoptotic multidomain Bcl-2 family proteins controls hepatocyte apoptosis in the healthy liver. The Bcl-2 homology domain 3 (BH3)-only proteins set this rheostatic balance toward apoptosis upon activation in the diseased liver. However, their involvement in healthy Bcl-2 rheostasis remains unknown. In the present study, we focused on two BH3-only proteins, Bim and Bid, and we clarified the Bcl-2 network that governs hepatocyte life and death in the healthy liver. We generated hepatocyte-specific Bcl-xL- or Mcl-1-knock-out mice, with or without disrupting Bim and/or Bid, and we examined hepatocyte apoptosis under physiological conditions. We also examined the effect of both Bid and Bim disruption on the hepatocyte apoptosis caused by the inhibition of Bcl-xL and Mcl-1. Spontaneous hepatocyte apoptosis in Bcl-xL- or Mcl-1-knock-out mice was significantly ameliorated by Bim deletion. The disruption of both Bim and Bid completely prevented hepatocyte apoptosis in Bcl-xL-knock-out mice and weakened massive hepatocyte apoptosis via the additional in vivo knockdown of mcl-1 in these mice. Finally, the hepatocyte apoptosis caused by ABT-737, which is a Bcl-xL/Bcl-2/Bcl-w inhibitor, was completely prevented in Bim/Bid double knock-out mice. The BH3-only proteins Bim and Bid are functionally active but are restrained by the anti-apoptotic Bcl-2 family proteins under physiological conditions. Hepatocyte integrity is maintained by the dynamic and well orchestrated Bcl-2 network in the healthy liver.

Keywords: Bcl-2 Family Proteins; Bcl-xl; Cell Biology; Hepatocyte; Liver Injury; Mcl-1; Mitochondrial Apoptosis.

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Figures

FIGURE 1.
FIGURE 1.
The disruption of Bim alleviated spontaneous hepatocyte apoptosis in the absence of Bcl-xL. A–F, the offspring from the mating of bim±bcl-xflox/floxalb-cre mice with bim±bcl-xflox/flox mice were examined at 6 weeks of age. Bcl-xL+/+ and Bcl-xL−/−, bcl-xflox/flox and bcl-xflox/floxalb-cre, respectively. A, Western blot analysis of whole liver lysates for the expression of BimEL, Bid, Bcl-xL, Mcl-1, Bak, Bax, Noxa, Bad, Puma, cleaved caspase-3, cleaved caspase-7, and β-actin. B, representative images for liver histology stained with hematoxylin-eosin (HE), TUNEL, and cleaved caspase-3 (original magnifications, ×100 (large panels) and ×400 (insets)); black arrows indicate apoptotic bodies. C, TUNEL-positive cell ratio; n = 8 mice/group; *, p < 0.05 versus all. D, cleaved caspase-3-positive cell ratio; n = 3 mice/group; *, p < 0.05 versus all. E, serum caspase-3/7 activity; n = 11 mice/group; *, p < 0.05 versus all. F, serum ALT levels; n = 13 mice/group; *, p < 0.05 versus all. G, offspring from the mating of bcl-xflox/floxalb-cre mice with bcl-xflox/flox mice were examined at 6 weeks of age. Bcl-xL+/+ and Bcl-xL−/−, bcl-xflox/flox and bcl-xflox/floxalb-cre, respectively. bim mRNA levels in the whole liver tissue were determined by real-time RT-PCR; n = 6 mice/group. Error bars, S.D. RLU, relative light units; I/U, international units.
FIGURE 2.
FIGURE 2.
The disruption of Bim alleviated spontaneous hepatocyte apoptosis in the absence of Mcl-1. The offspring from the mating of bim+−mcl-1flox/floxalb-cre mice with bim+/−mcl-1flox/flox mice were examined at 6 weeks of age. Mcl-1+/+ and Mcl-1−/−, mcl-1flox/flox and mcl-1flox/floxalb-cre, respectively. A, Western blot analysis of whole liver lysates for the expression of BimEL, Bid, Bcl-xL, Mcl-1, Bak, Bax, cleaved caspase-3, cleaved caspse-7, and β-actin. B, representative images for liver histology stained with hematoxylin-eosin (HE), TUNEL, and cleaved caspase-3 (original magnification, ×100). C, TUNEL-positive cell ratio; n = 3–6 mice/group; *, p < 0.05 versus all. D, cleaved caspase-3-positive cell ratio; n = 3 mice/group; *, p < 0.05 versus all. E, serum caspase-3/7 activity; n = 9–15 mice/group; *, p < 0.05 versus all. F, serum ALT levels; n = 9–15 mice/group; *, p < 0.05 versus all. Error bars, S.D. RLU, relative light units; I/U, international units.
FIGURE 3.
FIGURE 3.
The disruption of Bim and Bid prevented spontaneous hepatocyte apoptosis in the absence of Bcl-xL. The offspring from the mating of bim+/−bid−/−bcl-xflox/floxalb-cre mice with bim+/−bid−/−bcl-xflox/flox mice were examined at 6 weeks of age. Bcl-xL+/+ and Bcl-xL−/−, bcl-xflox/flox and bcl-xflox/floxalb-cre, respectively. A, Western blot analysis of whole liver lysates for the expression of BimEL, Bid, Bcl-xL, Mcl-1, Bak, Bax, and β-actin. B, representative images of liver histology stained with hematoxylin-eosin (HE) and TUNEL (original magnifications, ×100 (large panels) and ×400 (insets)). Black arrows indicate apoptotic bodies. C, TUNEL-positive cell ratio; more than 5 mice/group; *, p < 0.05 versus all. D, serum caspase-3/7 activity; more than 6 mice/group; *, p < 0.05 versus all. E, serum ALT levels; more than 6 mice/group; *, p < 0.05 versus all. Error bars, S.D. RLU, relative light units; I/U, international units.
FIGURE 4.
FIGURE 4.
Bim and Bid are essential regulators involved in the intrinsic pathway of apoptosis in hepatocytes in the absence of anti-apoptotic Bcl-2 family proteins. bcl-xflox/floxalb-cre mice and bim−/−bid−/−bcl-xflox/floxalb-cre mice were injected with mcl-1 or with negative control siRNA via the tail vein and were sacrificed 24 h (A and C–F) or 48 h (B) later. Bcl-xL+/+ and Bcl-xL−/−, bcl-xflox/flox and bcl-xflox/floxalb-cre, respectively. NC, negative control. A, Western blot analysis of whole liver lysates for the expression of BimEL, Bid, Bcl-xL, Mcl-1, Bak, Bax, and β-actin. B, representative images of liver histology stained with hematoxylin-eosin (original magnifications, ×100 (large panels) and ×400 (insets)). C, representative images of liver histology stained with TUNEL (original magnification, ×100). D, serum caspase-3/7 activity; n = 3–4 mice/group. E, serum ALT levels; n = 4 mice/group; data are presented as means ± S.E. (error bars). F, serum T-bilirubin levels; n = 4 mice/group. RLU, relative light units; I/U, international units.
FIGURE 5.
FIGURE 5.
The presence of Bim- and Bid-induced constant BH3 stress in the healthy liver causes hepatotoxicity with the use of anti-cancer agents that target anti-apoptotic Bcl-2 family proteins. The offspring from bim+/−bid−/− mating pairs were given an intraperitoneal injection of ABT-737 (100 mg/kg) or vehicle and were examined after 6 h. A, Western blot analysis of whole liver lysates for the expression of BimEL, Bid, Bcl-xL, Mcl-1, Bak, Bax, and β-actin. B and C, representative images of liver histology stained with hematoxylin-eosin and TUNEL (original magnifications, ×100 (large panels) and ×400 (insets)). D, TUNEL-positive cell ratio; n = 5–6 mice/group; *, p < 0.05 versus all. E, serum caspase-3/7 activity; more than 5 mice/group; *, p < 0.05 versus all. F, serum ALT levels; more than 5 mice/group; *, p < 0.05 versus all. Error bars, S.D. RLU, relative light units; I/U, international units.
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