Collaborative orchestration of BH3-only proteins governs Bak/Bax-dependent hepatocyte apoptosis under antiapoptotic protein-deficiency in mice.

The fine-tuned balance between anti-apoptotic Bcl-2 family proteins, such as Bcl-xL and Mcl-1, and pro-apoptotic Bcl-2 family proteins, like Bak and Bax, is crucial for maintaining hepatocyte integrity. BH3-only proteins, including Bid, Bim, Puma, Noxa, Bad, Bmf, Bik and Hrk, serve as apoptosis initiators. They are activated by various stimuli, which leads to Bak/Bax activation. We previously reported that Bid and Bim contributed to hepatocyte apoptosis through Bak/Bax activation in the absence of anti-apoptotic proteins Bcl-xL and/or Mcl-1. However, the comprehensive involvement of all eight BH3-only proteins in Bak/Bax-dependent hepatocyte apoptosis remains unclear. Puma disruption suppressed hepatocyte apoptosis in hepatocyte-specific Bcl-xL or Mcl-1 knockout (Bcl-xL(ΔHep/ΔHep) or Mcl-1(ΔHep/ΔHep)) mice. Disruption of Bid and Bim partially prevented lethality in Mcl-1(ΔHep/+) Bcl-xL(ΔHep/ΔHep) mice, although severe hepatocyte apoptosis persisted, which was suppressed by additional Puma disruption. However, hepatocyte apoptosis was still induced compared to that in Mcl-1(ΔHep/+) Bcl-xL(ΔHep/ΔHep) Bax(ΔHep/ΔHep) Bak(-/-) mice. Triple disruption of Bid, Bim and Puma did not prevent induction of hepatocyte apoptosis in tamoxifen-induced Mcl-1(iΔHep/iΔHep) Bcl-xL(iΔHep/iΔHep) mice. Primary hepatocytes, isolated from Mcl-1(fl/fl) Bcl-xL(fl/fl) Bid(-/-) Bim(-/-) Puma(-/-) mice and immortalized, underwent apoptosis with doxycycline-dependent Cre recombination. Among the remaining five BH3-only proteins, Bik and Hrk were not expressed in these cells, and Noxa knockdown, but not Bad or Bmf knockdown, reduced apoptosis. Noxa disruption alleviated hepatocyte apoptosis in Mcl-1(ΔHep/ΔHep) mice and tamoxifen-induced Mcl-1(iΔHep/iΔHep) Bcl-xL(iΔHep/iΔHep) Bid(-/-) Bim(-/-) Puma(-/-) mice, prolonging survival. Apoptosis persisted in immortalized primary hepatocytes isolated from Mcl-1(fl/fl) Bcl-xL(fl/fl) Bid(-/-) Bim(-/-) Puma(-/-) Noxa(-/-) mice where doxycycline-dependent Cre recombination was induced, but was completely suppressed by Bak/Bax knockdown, while Bad or Bmf knockdown had no effect. In conclusion, among the eight BH3-only proteins, Puma and Noxa, alongside Bid and Bim, contributed to Bak/Bax-dependent hepatocyte apoptosis, but not indispensably, in the absence of Mcl-1 and Bcl-xL.