Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Aug 9;294(32):11994-12006.
doi: 10.1074/jbc.RA118.007161. Epub 2019 Jun 19.

Interferon-γ induces the cell surface exposure of phosphatidylserine by activating the protein MLKL in the absence of caspase-8 activity

Jiancheng Chen  1 , Shunsuke Kuroki  2 , Masataka Someda  1 , Shin Yonehara  3
Affiliations

Interferon-γ induces the cell surface exposure of phosphatidylserine by activating the protein MLKL in the absence of caspase-8 activity

Jiancheng Chen et al. J Biol Chem. .

Abstract

Phosphatidylserine (PS), an anionic phospholipid enriched in the inner leaflet of the plasma membrane, is exposed to the outer leaflet during apoptosis. PS exposure was recently shown to be induced during tumor necrosis factor-induced necroptosis. We herein demonstrated that interferon (IFN)-γ induced necroptosis in Caspase-8-knockout mouse-derived embryonic fibroblasts (C8KO MEFs), as well as in WT MEFs co-treated with the pan-caspase inhibitor, z-VAD-fmk. PS exposure and necroptosis were significant after 6- and 24-h treatments with IFN-γ, respectively. To elucidate the molecular mechanisms underlying IFN-γ-induced PS exposure, we generated C8KO MEF-derived cell lines without the expression of RIPK3 (receptor-interacting protein kinase 3), an essential molecule in tumor necrosis factor-induced necroptosis, and IFN-γ-induced PS exposure and necrotic cell death were shown to be specifically inhibited by the loss of RIPK3 expression. Furthermore, the down-regulated expression of MLKL (mixed lineage kinase domain-like protein), a key molecule for inducing membrane rupture downstream of RIPK3 in necroptosis, abolished IFN-γ-induced PS exposure in C8KO MEFs. In human colorectal adenocarcinoma-derived HT29 cells, PS exposure and necroptosis were similarly induced by treatment with IFN-γ in the presence of Smac mimetics and z-VAD-fmk. The removal of IFN-γ from PS-exposing MEFs after a 6-h treatment completely inhibited necroptotic cell death but not the subsequent increase in the number of PS-exposing cells. Therefore, PS exposure mediated by RIPK3-activated MLKL oligomers was induced by a treatment with IFN-γ for a significant interval of time before the induction of necroptosis by membrane rupture.

Keywords: MLKL; RIPK3; caspase; cell death; interferon; interferon-gamma; necroptosis; necrosis (necrotic death); phosphatidylserine; receptor-interacting protein (RIP).

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
IFN-γ induces PS exposure in WT and C8KO MEFs before the induction of cell death. A and B, primary WT MEFs were treated with 10 ng/ml IFN-γ and 50 μm z-VAD-fmk (IZ) for the indicated periods. The cells were stained with MFG-E8-GFP and PI to monitor PS exposure and cell death, respectively, and then analyzed by fluorescent microscopy (A) and flow cytometry (B). BF, Brightfield. C, the quantified data of the flow cytometric analysis from B are graphically shown. D, primary C8KO MEFs were treated with 10 ng/ml IFN-γ for the indicated periods. The cells were stained with MFG-E8-GFP and PI and then analyzed by fluorescent microscopy. Representative fluorescent images and flow cytometric plots of more than three experiments are shown, and the scale bars represent 100 μm. The quantified data of the flow cytometric analysis are presented as means ± S.D. (n ≥ 3).
Figure 2.
Figure 2.
RIPK3 is necessary for IFN-γ–induced PS exposure in iC8KO MEFs. A, cell lysates from primary WT MEFs, primary C8KO MEFs, and iC8KO MEFs harboring the indicated expression vectors were analyzed by Western blotting with anti-RIPK3 and anti-actin antibodies. EV, an empty vector. *, nonspecific bands. B, iC8KO-RIPK3 MEFs were treated with 10 ng/ml IFN-γ for the indicated periods. The cells were stained with MFG-E8-GFP and PI and then analyzed by fluorescent microscopy. BF, Brightfield. The scale bars represent 100 μm. C, EV-expressing iC8KO (iC8KO-EV) and iC8KO-RIPK3 MEFs were treated with 10 ng/ml IFN-γ for the indicated periods. The cells were stained with MFG-E8-GFP and PI and analyzed by flow cytometry. D, the quantified data of the flow cytometric analysis from C are graphically shown. Representative Western blotting data, fluorescent images, and flow cytometric plots of more than three experiments are shown. The quantified data of the flow cytometric analysis are presented as means ± S.D. (n ≥ 3).
Figure 3.
Figure 3.
PS is exposed in a MLKL-dependent manner in IFN-γ–treated iC8KO-RIPK3 MEFs. A and B, iC8KO-RIPK3 MEFs were infected with lentiviral vectors encoding two different shRNAs for MLKL (shMlkl#1 and shMlkl#2). The expression levels of MLKL were analyzed by qRT-PCR (A) and by Western blotting with anti-MLKL and anti-actin antibodies (B). *, nonspecific bands. C, iC8KO-RIPK3 MEFs expressing shLacZ or shMlkl#2 were treated with 10 ng/ml IFN-γ for the indicated periods. The cells were stained with MFG-E8-GFP and PI and analyzed by flow cytometry. D, the quantified data of the flow cytometric analysis from C are graphically shown. Representative qRT-PCR data, Western blotting data, fluorescent images, and flow cytometric plots of more than three experiments are shown. For qRT-PCR analysis and flow cytometry quantification, the data are presented as means ± S.D. (n ≥ 3).
Figure 4.
Figure 4.
TMEM16F is dispensable for IFN-γ–induced PS exposure. A and B, iC8KO-RIPK3 MEFs were infected with a lentiviral vector encoding shRNA for TMEM16F (sh16F). The expression of TMEM16F was quantified by qRT-PCR (A) and Western blotting with anti-TMEM16F and anti-actin antibodies (B). *, nonspecific bands. C and D, iC8KO-RIPK3 MEFs expressing shLacZ or sh16F were treated with 10 ng/ml IFN-γ for the indicated periods. The cells were stained with MFG-E8-GFP and PI and analyzed by fluorescent microscopy (C) and flow cytometry (D). BF, Brightfield. The scale bar represents 100 μm. E, the quantified data of the flow cytometric analysis from D are graphically shown. Representative qRT-PCR data, Western blotting data, fluorescent images, and flow cytometric plots of more than three experiments are shown. For qRT-PCR analysis and flow cytometry quantification, the data are presented as means ± S.D. (n ≥ 3).
Figure 5.
Figure 5.
The MLKL protein forms a trimer in PS-exposing MEFs treated with IFN-γ. A and B, primary WT MEFs (A) and iC8KO-RIPK3 MEFs (B) were treated with 10 ng/ml IFN-γ and 50 μm z-VAD-fmk (IZ) and with 10 ng/ml IFN-γ, respectively, for the indicated periods. Cell lysates were resolved on reducing SDS-PAGE and analyzed by Western blotting with anti-phosphorylated MLKL (pMLKL), anti-MLKL, and anti-actin antibodies. C and D, cell lysates of primary WT MEFs treated with IZ (C) or with 10 ng/ml TNFα, 1 μg/ml cycloheximide, and 50 μm z-VAD-fmk (TCZ) (D) for the indicated periods were resolved on reducing or nonreducing SDS-PAGE and analyzed by Western blotting with anti-MLKL and anti-actin antibodies. E and F, iC8KO-RIPK3 MEFs (E) or primary WT MEFs, iC8KO-RIPK3 MEFs, and iC8KO-RIPK3 MEFs expressing shMlkl#2 (F) were treated with IFN-γ for the indicated periods. Cell lysates were resolved on reducing or nonreducing SDS-PAGE and analyzed by Western blotting with anti-MLKL and anti-actin antibodies. *, nonspecific bands. Representative Western blotting data of more than three experiments are shown.
Figure 6.
Figure 6.
Formation and shedding of membrane bubbles in IFN-γ–treated MEFs exposing PS. iC8KO-RIPK3 MEFs were treated with 10 ng/ml IFN-γ for the indicated periods and then directly stained with MFG-E8-GFP. The cells were then observed under fluorescent microscopy. The scale bars represent 10 μm. Representative fluorescent images of more than three experiments are shown. BF, Brightfield.
Figure 7.
Figure 7.
IFN-γ induces PS exposure without the induction of necroptotic cell death. A, after a 6-h treatment of iC8KO-RIPK3 MEFs with 10 ng/ml IFN-γ, IFN-γ was removed, and the cells were washed with prewarmed PBS twice and then cultured with fresh culture medium for the indicated periods. The cells at the indicated time points were stained with MFG-E8-GFP and PI and then analyzed by flow cytometry. B, the quantified data of the flow cytometric analysis from A are graphically shown. C, after a 6-h treatment of iC8KO-RIPK3 MEFs with 10 ng/ml IFN-γ, the cells were stained with MFG-E8-GFP and PI, and the MFG-E8-GFP+PI− cells were sorted and immediately analyzed by flow cytometry. Sorted cells were cultured without IFN-γ for 12 h or 2 days and analyzed by flow cytometry after staining with MFG-E8-GFP and PI. PS exposure by the sorted cells after cultivation without IFN-γ for 12 h and 2 days was again assessed by flow cytometry after a treatment with 10 ng/ml IFN-γ for 6 h. D, the quantified data of the flow cytometric analysis from C are graphically shown. Representative flow cytometric plots of more than three experiments are shown. For flow cytometry quantification, the data are presented as means ± S.D. (n ≥ 3).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Leventis P. A., and Grinstein S. (2010) The distribution and function of phosphatidylserine in cellular membranes. Annu. Rev. Biophys. 39, 407–427 10.1146/annurev.biophys.093008.131234 - DOI - PubMed
    1. Segawa K., and Nagata S. (2015) An apoptotic “eat me” signal: phosphatidylserine exposure. Trends Cell Biol. 25, 639–650 10.1016/j.tcb.2015.08.003 - DOI - PubMed
    1. Fadok V. A., Voelker D. R., Campbell P. A., Cohen J. J., Bratton D. L., and Henson P. M. (1992) Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J. Immunol. 148, 2207–2216 - PubMed
    1. Suzuki J., Imanishi E., and Nagata S. (2014) Exposure of phosphatidylserine by Xk-related protein family members during apoptosis. J. Biol. Chem. 289, 30257–30267 10.1074/jbc.M114.583419 - DOI - PMC - PubMed
    1. Suzuki J., Denning D. P., Imanishi E., Horvitz H. R., and Nagata S. (2013) Xk-related protein 8 and CED-8 promote phosphatidylserine exposure in apoptotic cells. Science 341, 403–406 10.1126/science.1236758 - DOI - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources