Disruption of Puma suppresses hepatocyte apoptosis in mice with hepatocyte-specific knockout of Bcl-xL or Mcl-1.
We initially examined the role of another BH3-only protein, Puma, in Bak/Bax activation in the absence of Bcl-xL or Mcl-1. We crossed Mcl-1ΔHep/ΔHep mice and Bcl-xLΔHep/ΔHep mice with Puma KO (Puma−/−) mice (Puma−/− mice), and generated Mcl-1ΔHep/Δhep Puma−/− mice and Bcl-xLΔhep/Δhep Puma−/− mice. The expression levels of Mcl-1 and Puma were reduced in the liver tissue of Mcl-1ΔHep/ΔHep Puma−/− mice (Fig. 1A). Similarly, Bcl-xL and Puma expression in the livers of Bcl-xLΔHep/ΔHep Puma−/− mice was reduced (Fig. 1B). Mcl-1ΔHep/ΔHep Puma−/− mice and Bcl-xLΔHep/ΔHep Puma−/− mice displayed significantly lower serum alanine transaminase (ALT) levels and reduced serum caspase 3/7 activity than Mcl-1ΔHep/ΔHep mice and Bcl-xLΔHep/ΔHep mice, respectively (Fig. 1C and 1D). These mice also showed a smaller number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-positive hepatocytes. (Fig. 1E and 1F).
Disruption of Bid, Bim and Puma cannot fully suppress hepatocyte apoptosis induced by the deletion of Bcl-xL and Mcl-1.
We have previously demonstrated that Bcl-xLΔHep/ΔHep Mcl-1ΔHep/+ mice, as well as Bcl-xLΔHep/ΔHep Mcl-1ΔHep/ΔHep mice, exhibit impaired liver development, leading to mortality within one day after birth10. Disruption of both Bid and Bim or Bid, Bim and Puma reduced the mortality of Bcl-xLΔHep/ΔHep Mcl-1ΔHep/+ mice but had no effect on that of Bcl-xLΔHep/ΔHep Mcl-1ΔHep/ ΔHep mice (Table 1, Table 2). Surviving Bcl-xLΔHep/ΔHep Mcl-1ΔHep/+ Bid−/− Bim−/− mice exhibited hepatocyte apoptosis with elevated serum ALT levels (Fig. 2A-2D). Further disruption of Puma significantly reduced the serum ALT levels, serum caspase 3/7 activity and number of TUNEL-positive hepatocytes in Bcl-xLΔHep/ΔHep Mcl-1ΔHep/+ Bid−/− Bim−/− mice (Fig. 2B-2D). However, the serum ALT levels in Bcl-xLΔHep/ΔHep Mcl-1ΔHep/+ Bid−/− Bim−/− Puma−/− mice were still significantly greater than those in Bcl-xLΔHep/ΔHep Mcl-1ΔHep/+ BaxΔHep/ΔHep Bak−/− mice (Fig. 2B).
We generated tamoxifen-inducible Bcl-xLiΔHep/iΔHep Mcl-1iΔHep/iΔHep Bid−/− Bim−/− Puma−/− mice, in which Bcl-xL and Mcl-1 expression are abolished upon tamoxifen injection (Fig. 2E), since the knockout of both Bcl-xL and Mcl-1 severely impairs liver development10 and no Bcl-xLΔHep/ΔHep Mcl-1ΔHep/ΔHep Bid−/− Bim−/− Puma−/− mice survive after birth (Table 2). After tamoxifen injection for three consecutive days, compared with control mice, Bcl-xLiΔHep/iΔHep Mcl-1iΔHep/iΔHep Bid−/− Bim−/− Puma−/− mice exhibited increased serum ALT levels, elevated caspase 3/7 activity and a higher number of TUNEL-positive hepatocytes (Fig. 2F-H). All mice in the Bcl-xLiΔHep/iΔHep Mcl-1iΔHep/iΔHep Bid−/− Bim−/− Puma−/− group were euthanized within 4 days after tamoxifen injection (data not shown). On the other hand, Bcl-xLiΔHep/iΔHep Mcl-1iΔHep/iΔHep BaxiΔHep/iΔHep Bak−/− mice showed no increase in serum ALT levels after tamoxifen injection compared to those without tamoxifen injection (Suppl. Figure 1A, 1B), and all of these mice survived to day 6 after tamoxifen injection (Suppl. Figure 1C). These findings suggested that another BH3-only protein, in addition to Bid, Bim and Puma, might activate Bak/Bax and induce hepatocyte apoptosis in the absence of both Bcl-xL and Mcl-1.
Noxa knockdown suppresses apoptosis in Bcl-xL-, Mcl-1-, Bid-, Bim- and Puma-deficient hepatocytes.
We generated a doxycycline-inducible Bcl-xLiΔHep/iΔHep Mcl-1iΔHep/iΔHep Bid−/− Bim−/− Puma−/− hepatocyte cell line using immortalized primary mouse hepatocytes whose Bcl-xL and Mcl-1 expression was abolished 48 hours after incubation with doxycycline (Fig. 3A). Caspase 3/7 activity and LDH activities were significantly increased, and the relative cell viability was significantly decreased after incubation with doxycycline (Fig. 3B). Among the five remaining BH3-only proteins, Noxa, Bad, and Bmf were expressed in this immortalized cell line, while Bik and Hrk were not detected (Fig. 3C). After doxycycline treatment, only Noxa expression increased among Noxa, Bad and Bmf (Fig. 3D). SiRNA-mediated knockdown of Noxa led to a significant reduction in Caspase 3/7 activity, LDH activities, and Annexin V-positive areas and a significant increase in the relative cell viability in doxycycline-treated Bcl-xLiΔHep/iΔHep Mcl-1iΔHep/iΔHep Bid−/− Bim−/− Puma−/− cells (Fig. 3E, 3H, Suppl. Figure 2A), whereas siRNA-mediated knockdown of Bad or Bmf did not (Fig. 3F, 3G, 3H, Suppl. Figure 2B, 2C).
Disruption of Noxa decreases hepatocyte apoptosis in Bcl-xL Mcl-1-, Bid-, Bim- and Puma-deficient mice and Mcl-1-deficient mice.
We disrupted Noxa using CRISPR-Cas9 technology to generate tamoxifen-inducible Bcl-xLiΔHep/iΔHep Mcl-1iΔHep/iΔHep Bid−/− Bim−/− Puma−/− Noxa−/− mice (Fig. 4A, Suppl. Figure 3A, 3B). Disruption of Noxa significantly reduced the serum ALT levels and caspase 3/7 activity in tamoxifen-treated Bcl-xLiΔHep/iΔHep Mcl-1iΔHep/iΔHep Bid−/− Bim−/− Puma−/ mice (Fig. 4B-4C). The number of TUNEL-positive hepatocytes was also significantly decreased by Noxa disruption (Fig. 4D). Although some Bcl-xLiΔHep/iΔHep Mcl-1iΔHep/iΔHep Bid−/− Bim−/− Puma−/− Noxa−/− mice died 5 days after tamoxifen injection, Noxa disruption significantly prolonged their survival (Fig. 4E).
To discern the potential contribution of Noxa to hepatocyte apoptosis, particularly in the presence of Bid, Bim and Puma, we knocked out Noxa in Mcl-1ΔHep/ΔHep mice (Fig. 5A). Compared with Noxa+/+ mice, Noxa−/− mice showed no differences in serum ALT levels, serum caspase 3/7 activity or number of TUNEL-positive cells (Fig. 5B-5D). Compared with Mcl-1ΔHep/ΔHep mice, Mcl-1ΔHep/ΔHep Noxa−/− mice exhibited significantly lower serum ALT levels and serum caspase 3/7 activity as well as fewer TUNEL-positive hepatocytes in liver sections (Fig. 5B-5D).
No BH3-only proteins other than Bid, Bim, Puma and Noxa contribute to hepatocyte apoptosis caused by the deletion of both Mcl-1 and Bcl-xL.
To examine the role of the remaining BH3-only proteins in hepatocyte apoptosis caused by the deletion of both Mcl-1 and Bcl-xL, we generated a doxycycline-inducible Bcl-xLiΔHep/iΔHep Mcl-1iΔHep/iΔHep Bid−/− Bim−/− Puma−/− Noxa−/− hepatocyte cell line using immortalized primary mouse hepatocytes. Caspase 3/7 activity and LDH activities were significantly increased, and the relative cell viability was significantly decreased after incubation with doxycycline (Fig. 6A, 6B). Among the four remaining BH3-only proteins, Bad and Bmf were expressed in Bcl-xLiΔHep/iΔHep Mcl-1iΔHep/iΔHep Bid−/− Bim−/− Puma−/− Noxa−/− cells. After doxycycline treatment, Bad and Bmf mRNA expression levels increased (Fig. 6C). However, siRNA-mediated knockdown of either of these genes did not affect caspase 3/7 activity, LDH activities or relative cell viability in doxycycline-treated Bcl-xLiΔHep/iΔHep Mcl-1iΔHep/iΔHep Bid−/− Bim−/− Puma−/− Noxa−/− cells (Fig. 6D, 6E). On the other hand, siRNA-mediated knockdown of both Bak and Bax completely abrogated the increases in caspase 3/7 activity and LDH activities and decreased cell viability (Fig. 6F-6H).
To explore other potential activators outside the realm of BH3-only proteins, we performed RNA-seq of a doxycycline-induced Bcl-xLiΔHep/iΔHep Mcl-1iΔHep/iΔHep Bid−/− Bim−/− Puma−/− Noxa−/− immortalized hepatocyte cell line and a doxycycline-induced Bcl-xLiΔHep/iΔHep Mcl-1iΔHep/iΔHep Bid−/− Bim−/− Puma−/− Noxa+/+ immortalized hepatocyte cell line (Fig. 3A, 6A). RNA-seq analysis revealed 977 genes in Bcl-xLiΔHep/iΔHep Mcl-1iΔHep/iΔHep Bid−/− Bim−/− Puma−/− Noxa−/− hepatocytes with FPKM values greater than twice those in Bcl-xLiΔHep/iΔHep Mcl-1iΔHep/iΔHep Bid−/− Bim−/− Puma−/− Noxa+/+ hepatocytes (Suppl. Table 1). Among these genes, no Bcl-2 family proteins were detected, while Caspase6, Caspase9 and Caspase12, which are associated with apoptosis11, 12, were detected. In the present study, we focused on caspase6, which is known as an executor caspase, as well as caspase3/caspase7. SiRNA-mediated knockdown of caspase6 slightly but significantly increased relative cell viability in doxycycline-treated Bcl-xLiΔHep/iΔHep Mcl-1iΔHep/iΔHep Bid−/− Bim−/− Puma−/− Noxa−/− cells, while it did not affect caspase-3/7 activity (Suppl. Figure 4A, 4B). Similarly, siRNA-mediated knockdown of caspase6 slightly increased the relative viability of BNL.CL.2 cells, a murine hepatocyte cell line, treated with ABT-737, a Bcl-xL inhibitor that induces Bak/Bax-dependent hepatocyte apoptosis13 without affecting caspase-3/7 activity (Suppl. Figure 5A).