Receptor interacting protein kinase 1 mediates murine acetaminophen toxicity independent of the necrosome and not through necroptosis

Abstract

Although necrosis in the acetaminophen (APAP) model is known to be regulated by c-Jun NH2-terminal kinase (JNK) through interaction with mitochondria, the role of necroptosis through receptor-interacting proteins 1 and 3 (RIPK1 and RIPK3) has also been suggested. Our aim was to determine the relationship between these two mechanisms of cell death. To verify the participation of RIPK1, we used antisense knockdown and confirmed protection comparable to the RIPK1 inhibitor, necrostatin, in vivo and in vitro. However, we found no evidence that RIPK3 is expressed in primary mouse hepatocytes under basal conditions or after APAP and RIPK3(-/-) mice were not protected. RIPK3 was exclusively expressed in nonparenchymal cells. RIPK1 knockdown protected RIPK3(-/-) mice to the same extent as wild-type mice, underscoring the independent role of RIPK1. We confirmed that necroptosis is not involved in APAP toxicity by using mixed lineage kinase domain-like protein (MLKL) knockout mice, which were not protected from APAP. Next, we addressed whether there is interplay between RIPK1 and JNK. RIPK1 knockdown decreased the level of JNK activation and translocation to mitochondria and abrogated subsequent translocation of dynamin-related protein 1 (Drp1). Interestingly, APAP induced translocation of RIPK1 to mitochondria, which was unaffected by knockdown of the mitochondrial JNK docking protein, Sh3 homology 3 binding protein 5 (Sab).

Conclusion: RIPK1 participates in APAP-induced necrosis upstream of JNK activation whereas RIPK3 and MLKL are dispensable, indicating that necroptosis does not contribute to APAP-induced necrosis and RIPK1 has a unique, independent role.

Figure 1. Effect of RIPK1 knockdown on APAP toxicity in vivo

Mice were treated with RIPK1 or Control ASO (50 mg/kg) five times, subsequently injected with APAP 300 mg/kg and euthanized at 24 hrs. (A) Serum ALT (U/L), (inset, Western blot showing protein knockdown). N=12 per group; * p value ≤ 0.05 RIPK1 vs control ASO treated mice. (B) Representative histology H&E (10x); (C) Western blot of NAPQI adducts and densitometry. N=3; (D) Mice were treated with RIPK1 or control ASO and subsequently injected with 500 mg/kg APAP and followed for 72 hrs to assess survival. Results of two independent experiments N=8 per group; RIPK1 Log Rank P. value <0.001.

Figure 1. Effect of RIPK1 knockdown on APAP toxicity in vivo

Mice were treated with RIPK1 or Control ASO (50 mg/kg) five times, subsequently injected with APAP 300 mg/kg and euthanized at 24 hrs. (A) Serum ALT (U/L), (inset, Western blot showing protein knockdown). N=12 per group; * p value ≤ 0.05 RIPK1 vs control ASO treated mice. (B) Representative histology H&E (10x); (C) Western blot of NAPQI adducts and densitometry. N=3; (D) Mice were treated with RIPK1 or control ASO and subsequently injected with 500 mg/kg APAP and followed for 72 hrs to assess survival. Results of two independent experiments N=8 per group; RIPK1 Log Rank P. value <0.001.

Figure 1. Effect of RIPK1 knockdown on APAP toxicity in vivo

Mice were treated with RIPK1 or Control ASO (50 mg/kg) five times, subsequently injected with APAP 300 mg/kg and euthanized at 24 hrs. (A) Serum ALT (U/L), (inset, Western blot showing protein knockdown). N=12 per group; * p value ≤ 0.05 RIPK1 vs control ASO treated mice. (B) Representative histology H&E (10x); (C) Western blot of NAPQI adducts and densitometry. N=3; (D) Mice were treated with RIPK1 or control ASO and subsequently injected with 500 mg/kg APAP and followed for 72 hrs to assess survival. Results of two independent experiments N=8 per group; RIPK1 Log Rank P. value <0.001.

Figure 2. RIPK1 translocates to mitochondria

(A) Mice were treated with RIPK1 or control ASO and subsequently injected with 300 mg/kg of APAP and euthanized at the indicated time points. Western blot of cytoplasmic fraction for RIPK1 and densitometry; (B) Western blot of mitochondrial fraction for RIPK1 and densitometry; N= 3 mice per time point per group. * p value ≤ 0.05 APAP vs control #, p value ≤ 0.05 RIPK1 ASO vs control ASO. (C) Co-localization of RIPK1 and COXIV in PMH after PBS vs APAP10 mM for 2 hrs (64X magnification). Green: cytochrome oxidase IV, red: RIPK1, yellow: co-localization, and blue: DAPI nuclear stain.

Figure 2. RIPK1 translocates to mitochondria

(A) Mice were treated with RIPK1 or control ASO and subsequently injected with 300 mg/kg of APAP and euthanized at the indicated time points. Western blot of cytoplasmic fraction for RIPK1 and densitometry; (B) Western blot of mitochondrial fraction for RIPK1 and densitometry; N= 3 mice per time point per group. * p value ≤ 0.05 APAP vs control #, p value ≤ 0.05 RIPK1 ASO vs control ASO. (C) Co-localization of RIPK1 and COXIV in PMH after PBS vs APAP10 mM for 2 hrs (64X magnification). Green: cytochrome oxidase IV, red: RIPK1, yellow: co-localization, and blue: DAPI nuclear stain.

Figure 3. Expression of RIPK3 in liver cells

WT mice were treated with PBS or APAP 300 mg/kg for 3 and 6 hours and RIPK3 −/− mice were treated with APAP for 6 hrs. (A) Immunoblot of 30 ug of whole liver lysate using Genentech RIPK3 monoclonal antibody at indicated time points and positive and negative controls and densitometry normalized to β-actin. (B) Immunoblot depicting liver cell fractions and positive (lane 1) and negative controls (lane2) for RIPK3. 50 ug of freshly isolated untreated PMH (lane 3) or treated with APAP 10 mM for 6 hr prior to Western blot (lane 4), 15 ug of Kupffer cells isolated from control (lane 5) or APAP (300 mg/kg) treated mice for 3 hr (lane 6), 15 ug of LSECs isolated from control mice using elutriation (lane 7), 10 ug total liver leukocyte (lane 8) and 10 ug of wild type spleen (lane 9). Ponceau stain for protein loading shown for comparison, GAPDH is loading for PMH Con vs APAP 6 hr. NS: nonspecific.

Figure 3. Expression of RIPK3 in liver cells

WT mice were treated with PBS or APAP 300 mg/kg for 3 and 6 hours and RIPK3 −/− mice were treated with APAP for 6 hrs. (A) Immunoblot of 30 ug of whole liver lysate using Genentech RIPK3 monoclonal antibody at indicated time points and positive and negative controls and densitometry normalized to β-actin. (B) Immunoblot depicting liver cell fractions and positive (lane 1) and negative controls (lane2) for RIPK3. 50 ug of freshly isolated untreated PMH (lane 3) or treated with APAP 10 mM for 6 hr prior to Western blot (lane 4), 15 ug of Kupffer cells isolated from control (lane 5) or APAP (300 mg/kg) treated mice for 3 hr (lane 6), 15 ug of LSECs isolated from control mice using elutriation (lane 7), 10 ug total liver leukocyte (lane 8) and 10 ug of wild type spleen (lane 9). Ponceau stain for protein loading shown for comparison, GAPDH is loading for PMH Con vs APAP 6 hr. NS: nonspecific.

Figure 4. Effect of RIPK1 knockdown and inhibition on RIPK3 −/− mice and hepatocytes

(A) Overnight fasted WT and RIPK3 −/− mice were treated with APAP 300 mg/kg and euthanized after 6 hr. N= 12 per group, ALT (U/L); (B) RIPK3−/− and WT controls were given Control ASO or RIPK1 ASO for five doses and then injected with 300 mg/kg of APAP and euthanized at 24 hrs, ALT (U/L), *P<0.05 RIPK1 vs Control ASO; (C) Representative histology (H&E) at 24 hr time point (10x). Results are mean ± S.D, For B&C N=12 at least per group; (D) Viability of PMH from WT vs RIPK3 −/− mice treated with APAP 20 mM for 2 hours. Subsequently media was exchanged and Nec-1 or DMSO was added. Results are mean ± S.D, N=3 *P<0.05.

Figure 4. Effect of RIPK1 knockdown and inhibition on RIPK3 −/− mice and hepatocytes

(A) Overnight fasted WT and RIPK3 −/− mice were treated with APAP 300 mg/kg and euthanized after 6 hr. N= 12 per group, ALT (U/L); (B) RIPK3−/− and WT controls were given Control ASO or RIPK1 ASO for five doses and then injected with 300 mg/kg of APAP and euthanized at 24 hrs, ALT (U/L), *P<0.05 RIPK1 vs Control ASO; (C) Representative histology (H&E) at 24 hr time point (10x). Results are mean ± S.D, For B&C N=12 at least per group; (D) Viability of PMH from WT vs RIPK3 −/− mice treated with APAP 20 mM for 2 hours. Subsequently media was exchanged and Nec-1 or DMSO was added. Results are mean ± S.D, N=3 *P<0.05.

Figure 4. Effect of RIPK1 knockdown and inhibition on RIPK3 −/− mice and hepatocytes

(A) Overnight fasted WT and RIPK3 −/− mice were treated with APAP 300 mg/kg and euthanized after 6 hr. N= 12 per group, ALT (U/L); (B) RIPK3−/− and WT controls were given Control ASO or RIPK1 ASO for five doses and then injected with 300 mg/kg of APAP and euthanized at 24 hrs, ALT (U/L), *P<0.05 RIPK1 vs Control ASO; (C) Representative histology (H&E) at 24 hr time point (10x). Results are mean ± S.D, For B&C N=12 at least per group; (D) Viability of PMH from WT vs RIPK3 −/− mice treated with APAP 20 mM for 2 hours. Subsequently media was exchanged and Nec-1 or DMSO was added. Results are mean ± S.D, N=3 *P<0.05.

Figure 4. Effect of RIPK1 knockdown and inhibition on RIPK3 −/− mice and hepatocytes

(A) Overnight fasted WT and RIPK3 −/− mice were treated with APAP 300 mg/kg and euthanized after 6 hr. N= 12 per group, ALT (U/L); (B) RIPK3−/− and WT controls were given Control ASO or RIPK1 ASO for five doses and then injected with 300 mg/kg of APAP and euthanized at 24 hrs, ALT (U/L), *P<0.05 RIPK1 vs Control ASO; (C) Representative histology (H&E) at 24 hr time point (10x). Results are mean ± S.D, For B&C N=12 at least per group; (D) Viability of PMH from WT vs RIPK3 −/− mice treated with APAP 20 mM for 2 hours. Subsequently media was exchanged and Nec-1 or DMSO was added. Results are mean ± S.D, N=3 *P<0.05.

Figure 5. MLKL expression in liver cells and APAP toxicity in MLKL −/−

(A) Immunoblot of MLKL, β-actin and GAPDH of liver cell fractions and BALB fibroblast cell lysate with variable loading. Positive control (lane 1, 10 ug) and negative control (lane2, 50 ug) for MLKL. Freshly isolated untreated PMH (lane 3, 30 ug) or treated with APAP 10 mM for 6 hr in vitro prior to Western blot (lane 4), Kupffer cells isolated from control (lane 5, 10 ug) or APAP (300 mg/kg) treated mice for 3 hr (lane 6, 10 ug), LSECs isolated from control mice using elutriation (lane 7, 10 ug), total liver leukocyte (lane 8, 5 ug), wild type spleen (lane 9, 5 ug) and MLKL −/− spleen (lane 10, 15 ug). Ponceau stain for protein loading shown for comparison among different cells. (B) Overnight fasted WT and MLKL −/− mice were treated with APAP 300 mg/kg and euthanized at 24 hrs, ALT (U/L) (inset, Western blot showing protein knockout) Results are mean ± S.D, N= 5 per group; (C) Representative histology (H&E) at 24 hrs (10X).

Figure 5. MLKL expression in liver cells and APAP toxicity in MLKL −/−

(A) Immunoblot of MLKL, β-actin and GAPDH of liver cell fractions and BALB fibroblast cell lysate with variable loading. Positive control (lane 1, 10 ug) and negative control (lane2, 50 ug) for MLKL. Freshly isolated untreated PMH (lane 3, 30 ug) or treated with APAP 10 mM for 6 hr in vitro prior to Western blot (lane 4), Kupffer cells isolated from control (lane 5, 10 ug) or APAP (300 mg/kg) treated mice for 3 hr (lane 6, 10 ug), LSECs isolated from control mice using elutriation (lane 7, 10 ug), total liver leukocyte (lane 8, 5 ug), wild type spleen (lane 9, 5 ug) and MLKL −/− spleen (lane 10, 15 ug). Ponceau stain for protein loading shown for comparison among different cells. (B) Overnight fasted WT and MLKL −/− mice were treated with APAP 300 mg/kg and euthanized at 24 hrs, ALT (U/L) (inset, Western blot showing protein knockout) Results are mean ± S.D, N= 5 per group; (C) Representative histology (H&E) at 24 hrs (10X).

Figure 6. Relation of RIPK1 with the activation of JNK and tranlocation of DRP1

Mice were treated with RIPK1 or control ASO and subsequently injected with 300 mg/kg of APAP and euthanized at the indicated time points; (A) Western blot of cytoplasm for P-JNK, total JNK, and β-actin and densitometry; (B) Western blot of mitochondrial fraction for p-JNK and PHB1 and densitometry; (C) Western blot of mitochondrial fraction of Control and RIPK1 ASO treated mice for Drp1 and densitometry; N= 3 mice per time point per group.* p value < 0.05 APAP vs control, # p value < 0.05 RIPK1 ASO vs control ASO; (D) Western blot of mitochondrial fraction of adenovirus shSab vs shLacZ treated mice for Drp1. (E) Western blot of mitochondrial fraction of adenovirus shSab vs shLacZ treated mice for RIPK1.

Figure 6. Relation of RIPK1 with the activation of JNK and tranlocation of DRP1

Mice were treated with RIPK1 or control ASO and subsequently injected with 300 mg/kg of APAP and euthanized at the indicated time points; (A) Western blot of cytoplasm for P-JNK, total JNK, and β-actin and densitometry; (B) Western blot of mitochondrial fraction for p-JNK and PHB1 and densitometry; (C) Western blot of mitochondrial fraction of Control and RIPK1 ASO treated mice for Drp1 and densitometry; N= 3 mice per time point per group.* p value < 0.05 APAP vs control, # p value < 0.05 RIPK1 ASO vs control ASO; (D) Western blot of mitochondrial fraction of adenovirus shSab vs shLacZ treated mice for Drp1. (E) Western blot of mitochondrial fraction of adenovirus shSab vs shLacZ treated mice for RIPK1.

Figure 6. Relation of RIPK1 with the activation of JNK and tranlocation of DRP1

Mice were treated with RIPK1 or control ASO and subsequently injected with 300 mg/kg of APAP and euthanized at the indicated time points; (A) Western blot of cytoplasm for P-JNK, total JNK, and β-actin and densitometry; (B) Western blot of mitochondrial fraction for p-JNK and PHB1 and densitometry; (C) Western blot of mitochondrial fraction of Control and RIPK1 ASO treated mice for Drp1 and densitometry; N= 3 mice per time point per group.* p value < 0.05 APAP vs control, # p value < 0.05 RIPK1 ASO vs control ASO; (D) Western blot of mitochondrial fraction of adenovirus shSab vs shLacZ treated mice for Drp1. (E) Western blot of mitochondrial fraction of adenovirus shSab vs shLacZ treated mice for RIPK1.

Figure 6. Relation of RIPK1 with the activation of JNK and tranlocation of DRP1

Mice were treated with RIPK1 or control ASO and subsequently injected with 300 mg/kg of APAP and euthanized at the indicated time points; (A) Western blot of cytoplasm for P-JNK, total JNK, and β-actin and densitometry; (B) Western blot of mitochondrial fraction for p-JNK and PHB1 and densitometry; (C) Western blot of mitochondrial fraction of Control and RIPK1 ASO treated mice for Drp1 and densitometry; N= 3 mice per time point per group.* p value < 0.05 APAP vs control, # p value < 0.05 RIPK1 ASO vs control ASO; (D) Western blot of mitochondrial fraction of adenovirus shSab vs shLacZ treated mice for Drp1. (E) Western blot of mitochondrial fraction of adenovirus shSab vs shLacZ treated mice for RIPK1.