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DNase II activated by the mitochondrial apoptotic pathway regulates RIP1-dependent non-apoptotic hepatocyte death via the TLR9/IFN-β signaling pathway

Cell Death & Differentiationvolume 26pages470486 (2019) | Download Citation

Abstract

Cell death, including apoptotic and non-apoptotic cell death, is frequently observed in liver disease. Upon activation of the mitochondrial apoptotic pathway, mitochondria release not only apoptogenic cytochrome c but also mitochondrial DNA (mtDNA) into the cytosol. The impact of DNase II, a lysosomal acid DNase that degrades mtDNA, on hepatocyte death remains unclear. Administration of ABT-737, a Bcl-xL inhibitor, upregulated DNase II activity in murine hepatocyte cell line BNL CL.2 cells and induced apoptosis. In cells treated with DNase II siRNA, ABT-737 led to accumulation of mtDNA in the cytosol and increased expression of interferon (IFN)-β and induction of propidium iodide (PI)-positive cells, in addition to apoptosis. Induced PI-positive cells were suppressed by RIP1 inhibitor, Necrostatin-1, but not by pan-caspase inhibitor, ZVAD-FMK, suggesting non-apoptotic cell death. Both the increase in IFN-β and the induction of non-apoptotic cell death were abolished by administering a TLR9 antagonist, ODN2088, or by the removal of mtDNA from cells with ethidium bromide. Hepatocyte-specific Mcl-1 knockout mice developed hepatocyte apoptosis accompanied by upregulated DNase II activity in their livers. Further knockout of DNase II induced IFN-β expression and RIP1-dependent non-apoptotic hepatocyte death, both of which were suppressed by the administration of ODN2088. Mice fed a high-fat diet (HFD), an obesity-associated fatty liver model, showed increased expression of IFN-β with suppression of DNase II activity in their livers and developed not only hepatocyte apoptosis but also non-apoptotic hepatocyte death. Hepatocyte-specific knockout of DNase II exacerbated HFD-induced non-apoptotic hepatocyte death and liver fibrosis. In conclusion, without DNase II, apoptotic stimulation on hepatocytes induces TLR9-dependent IFN-β production and RIP1-dependent non-apoptotic cell death originating from mtDNA. In fatty livers, DNase II activity is suppressed in contrast to simple inactivation of Bcl-xL or Mcl-1, and both apoptotic and non-apoptotic hepatocyte death can develop, leading to the progression of liver fibrosis.

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Edited by H.-U. Simon

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Acknowledgements

We sincerely thank AbbVie for providing ABT-737, Dr. S. Nagata (Kyoto University) for providing Dnase2a floxed mice, Dr. You-Wen He (Department of Immunology, Duke University Medical Center, Durham, NC) for providing the Mcl-1 floxed mice and Dr. Kinya Otsu (King’s College London) and Dr. Osamu Yamaguchi (Osaka University) for technical assistance with the measurement of DNase II activity using the SRED method.

Funding

This work was partially supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (26253047 and 17H05508 to T Takehara and 17K09423 to HH) and a Grant-in-Aid for Research on Hepatitis from the Ministry of Health, Labour and Welfare of Japan.

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  1. Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Osaka, Japan

    • Yoshinobu Saito
    • , Hayato Hikita
    • , Yasutoshi Nozaki
    • , Yugo Kai
    • , Yuki Makino
    • , Tasuku Nakabori
    • , Satoshi Tanaka
    • , Ryoko Yamada
    • , Minoru Shigekawa
    • , Takahiro Kodama
    • , Ryotaro Sakamori
    • , Tomohide Tatsumi
    •  & Tetsuo Takehara

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The authors declare that they have no conflict of interest.

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Correspondence to Tetsuo Takehara.

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DOI

https://doi.org/10.1038/s41418-018-0131-6