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NEMO Prevents Steatohepatitis and Hepatocellular Carcinoma by Inhibiting RIPK1 Kinase Activity-Mediated Hepatocyte Apoptosis.

Kondylis V, Polykratis A, Ehlken H, Ochoa-Callejero L, Straub BK, Krishna-Subramanian S, Van TM, Curth HM, Heise N, Weih F, Klein U, Schirmacher P, Kelliher M, Pasparakis M - Cancer Cell (2015)

Bottom Line: Mice lacking NEMO in liver parenchymal cells (LPC) spontaneously develop steatohepatitis and hepatocellular carcinoma (HCC) suggesting that NF-κB prevents liver disease and cancer.Here, we show that complete NF-κB inhibition by combined LPC-specific ablation of RelA, c-Rel, and RelB did not phenocopy NEMO deficiency, but constitutively active IKK2-mediated NF-κB activation prevented hepatocellular damage and HCC in NEMO(LPC-KO) mice.Knock-in expression of kinase inactive receptor-interacting protein kinase 1 (RIPK1) prevented hepatocyte apoptosis and HCC, while RIPK1 ablation induced TNFR1-associated death domain protein (TRADD)-dependent hepatocyte apoptosis and liver tumors in NEMO(LPC-KO) mice, revealing distinct kinase-dependent and scaffolding functions of RIPK1.

View Article: PubMed Central - PubMed

Affiliation: Institute for Genetics, University of Cologne, 50674 Cologne, Germany; Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, 50931 Cologne, Germany; Center for Molecular Medicine (CMMC), University of Cologne, 50931, Cologne, Germany.

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Lack of RIPK1 Kinase Activity, but Not Its Scaffolding Function, Prevents Hepatocyte Apoptosis in NEMOLPC-KO Mice(A) Serum ALT levels in 8-week-old mice with the indicated genotypes. Horizontal lines indicate mean values.(B) Immunoblot analysis of liver lysates from 8-week-old mice with the indicated genotypes. Actin was used as loading control.(C) Representative images of liver sections from 8-week-old mice with the indicated genotypes after staining with H&E or immunostaining with the indicated antibodies. Graphs depict quantification of the indicated parameters (mean ± SD, n = 3–5 mice per genotype). Scale bars, 200 μm.See also Figure S5.
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fig5: Lack of RIPK1 Kinase Activity, but Not Its Scaffolding Function, Prevents Hepatocyte Apoptosis in NEMOLPC-KO Mice(A) Serum ALT levels in 8-week-old mice with the indicated genotypes. Horizontal lines indicate mean values.(B) Immunoblot analysis of liver lysates from 8-week-old mice with the indicated genotypes. Actin was used as loading control.(C) Representative images of liver sections from 8-week-old mice with the indicated genotypes after staining with H&E or immunostaining with the indicated antibodies. Graphs depict quantification of the indicated parameters (mean ± SD, n = 3–5 mice per genotype). Scale bars, 200 μm.See also Figure S5.

Mentions: Inhibition of RIPK1 kinase activity by necrostatin-1 or other small molecule inhibitors prevents RIPK3/MLKL-dependent necroptosis and under certain conditions apoptosis (Christofferson et al., 2014, Pasparakis and Vandenabeele, 2015, Vanden Berghe et al., 2014). To address the role of RIPK1 kinase activity in hepatocyte death and HCC development in NEMOLPC-KO mice, we crossed them to knock-in mice expressing a kinase-inactive mutant RIPK1 (RIPK1D138N) (Polykratis et al., 2014). Strikingly, 8-week-old NEMOLPC-KO;Ripk1D138N/D138N mice showed strongly reduced serum ALT levels compared to NEMOLPC-KO mice (Figure 5A). Immunoblot analysis showed lack of caspase-3 and JNK activation in liver extracts from 8-week-old NEMOLPC-KO;Ripk1D138N/D138N mice (Figure 5B). Accordingly, histological analysis of livers from NEMOLPC-KO;Ripk1D138N/D138N mice showed significantly reduced hepatocyte apoptosis and proliferation and HSC activation compared to NEMOLPC-KO mice (Figure 5C). To exclude the possibility that inhibition of RIPK1 kinase activity in a cell type different from LPCs was responsible for the protective effect, we generated NEMOLPC-KO mice carrying one loxP-flanked Ripk1 allele and one Ripk1D138N allele. In these mice, Alfp-Cre-mediated deletion of the floxed Ripk1 allele results in expression of only the RIPK1D138N protein in LPCs, while all other cells express both wild-type RIPK1 and the mutant RIPK1D138N protein. Similarly to NEMOLPC-KO;Ripk1D138N/D138N mice, these NEMOLPC-KO;RIPK1LPC-KO/D138N mice showed strongly reduced liver damage (Figure 5A and data not shown). Collectively, these results showed that lack of RIPK1 kinase activity strongly protected NEMO-deficient hepatocytes from apoptosis.


NEMO Prevents Steatohepatitis and Hepatocellular Carcinoma by Inhibiting RIPK1 Kinase Activity-Mediated Hepatocyte Apoptosis.

Kondylis V, Polykratis A, Ehlken H, Ochoa-Callejero L, Straub BK, Krishna-Subramanian S, Van TM, Curth HM, Heise N, Weih F, Klein U, Schirmacher P, Kelliher M, Pasparakis M - Cancer Cell (2015)

Lack of RIPK1 Kinase Activity, but Not Its Scaffolding Function, Prevents Hepatocyte Apoptosis in NEMOLPC-KO Mice(A) Serum ALT levels in 8-week-old mice with the indicated genotypes. Horizontal lines indicate mean values.(B) Immunoblot analysis of liver lysates from 8-week-old mice with the indicated genotypes. Actin was used as loading control.(C) Representative images of liver sections from 8-week-old mice with the indicated genotypes after staining with H&E or immunostaining with the indicated antibodies. Graphs depict quantification of the indicated parameters (mean ± SD, n = 3–5 mice per genotype). Scale bars, 200 μm.See also Figure S5.
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fig5: Lack of RIPK1 Kinase Activity, but Not Its Scaffolding Function, Prevents Hepatocyte Apoptosis in NEMOLPC-KO Mice(A) Serum ALT levels in 8-week-old mice with the indicated genotypes. Horizontal lines indicate mean values.(B) Immunoblot analysis of liver lysates from 8-week-old mice with the indicated genotypes. Actin was used as loading control.(C) Representative images of liver sections from 8-week-old mice with the indicated genotypes after staining with H&E or immunostaining with the indicated antibodies. Graphs depict quantification of the indicated parameters (mean ± SD, n = 3–5 mice per genotype). Scale bars, 200 μm.See also Figure S5.
Mentions: Inhibition of RIPK1 kinase activity by necrostatin-1 or other small molecule inhibitors prevents RIPK3/MLKL-dependent necroptosis and under certain conditions apoptosis (Christofferson et al., 2014, Pasparakis and Vandenabeele, 2015, Vanden Berghe et al., 2014). To address the role of RIPK1 kinase activity in hepatocyte death and HCC development in NEMOLPC-KO mice, we crossed them to knock-in mice expressing a kinase-inactive mutant RIPK1 (RIPK1D138N) (Polykratis et al., 2014). Strikingly, 8-week-old NEMOLPC-KO;Ripk1D138N/D138N mice showed strongly reduced serum ALT levels compared to NEMOLPC-KO mice (Figure 5A). Immunoblot analysis showed lack of caspase-3 and JNK activation in liver extracts from 8-week-old NEMOLPC-KO;Ripk1D138N/D138N mice (Figure 5B). Accordingly, histological analysis of livers from NEMOLPC-KO;Ripk1D138N/D138N mice showed significantly reduced hepatocyte apoptosis and proliferation and HSC activation compared to NEMOLPC-KO mice (Figure 5C). To exclude the possibility that inhibition of RIPK1 kinase activity in a cell type different from LPCs was responsible for the protective effect, we generated NEMOLPC-KO mice carrying one loxP-flanked Ripk1 allele and one Ripk1D138N allele. In these mice, Alfp-Cre-mediated deletion of the floxed Ripk1 allele results in expression of only the RIPK1D138N protein in LPCs, while all other cells express both wild-type RIPK1 and the mutant RIPK1D138N protein. Similarly to NEMOLPC-KO;Ripk1D138N/D138N mice, these NEMOLPC-KO;RIPK1LPC-KO/D138N mice showed strongly reduced liver damage (Figure 5A and data not shown). Collectively, these results showed that lack of RIPK1 kinase activity strongly protected NEMO-deficient hepatocytes from apoptosis.

Bottom Line: Mice lacking NEMO in liver parenchymal cells (LPC) spontaneously develop steatohepatitis and hepatocellular carcinoma (HCC) suggesting that NF-κB prevents liver disease and cancer.Here, we show that complete NF-κB inhibition by combined LPC-specific ablation of RelA, c-Rel, and RelB did not phenocopy NEMO deficiency, but constitutively active IKK2-mediated NF-κB activation prevented hepatocellular damage and HCC in NEMO(LPC-KO) mice.Knock-in expression of kinase inactive receptor-interacting protein kinase 1 (RIPK1) prevented hepatocyte apoptosis and HCC, while RIPK1 ablation induced TNFR1-associated death domain protein (TRADD)-dependent hepatocyte apoptosis and liver tumors in NEMO(LPC-KO) mice, revealing distinct kinase-dependent and scaffolding functions of RIPK1.

View Article: PubMed Central - PubMed

Affiliation: Institute for Genetics, University of Cologne, 50674 Cologne, Germany; Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, 50931 Cologne, Germany; Center for Molecular Medicine (CMMC), University of Cologne, 50931, Cologne, Germany.

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