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, Bik, Bmf and Hrk, serve as apoptosis sensors activating Bak and Bax. We previously reported that BH3-only proteins Bid and Bim contribute to hepatocyte apoptosis through Bak/Bax activation in the absence of antiapoptotic proteins, Bcl-xL and/or Mcl-1. However, the comprehensive involvement of all eight BH3-only proteins maintaining hepatocyte integrity in healthy livers 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 strongly 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 this cell line, 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 sustained Bak/Bax-dependent hepatocyte apoptosis in the absence of Mcl-1 and Bcl-xL, elucidating the orchestration of Bcl-2 family proteins in healthy livers.