Cysteine protease cathepsin B mediates radiation-induced bystander effects

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Abstract

The radiation-induced bystander effect (RIBE) refers to a unique process in which factors released by irradiated cells or tissues exert effects on other parts of the animal not exposed to radiation, causing genomic instability, stress responses and altered apoptosis or cell proliferation1,2,3. Although RIBEs have important implications for radioprotection, radiation safety and radiotherapy, the molecular identities of RIBE factors and their mechanisms of action remain poorly understood. Here we use Caenorhabditis elegans as a model in which to study RIBEs, and identify the cysteine protease CPR-4, a homologue of human cathepsin B, as the first RIBE factor in nematodes, to our knowledge. CPR-4 is secreted from animals irradiated with ultraviolet or ionizing gamma rays, and is the major factor in the conditioned medium that leads to the inhibition of cell death and increased embryonic lethality in unirradiated animals. Moreover, CPR-4 causes these effects and stress responses at unexposed sites distal to the irradiated tissue. The activity of CPR-4 is regulated by the p53 homologue CEP-1 in response to radiation, and CPR-4 seems to exert RIBEs by acting through the insulin-like growth factor receptor DAF-2. Our study provides crucial insights into RIBEs, and will facilitate the identification of additional RIBE factors and their mechanisms of action.

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Figure 1: Identification of the RIBE factor.
Figure 2: CPR-4 is a RIBE factor.
Figure 3: CPR-4 and DAF-2 mediate RIBE in a localized UV irradiation model.
Figure 4: CPR-4 acts through DAF-2 to exert RIBE.

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Acknowledgements

We thank J. Tyler for help with localized irradiation experiments and T. Su for discussion. This work was supported by National Basic Research Program of China (2013CB945602), National Scientific and Technological Major Project of China (2013ZX10002-002), fellowships from China Scholarship Council and Fujian Agriculture and Forestry University (L.Z.) and Tsinghua University-Peking University Center for Life Sciences (Q.L. and X.Z.), and NIH grant R35 GM118188 (D.X.).

Author information

Y.P. set up conditioned medium RIBE assays and performed fractionation, RNAi screen, protein purification, and protease assays. M.Z. and Q.L. generated transgenic animals and conducted experiments with Y.P., aided by H.G. and X.Z. H.L. and L.Z. set up intra-animal RIBE assays and collected related data, aided by Y.-Z.C., X.W. and B.L. J.-T.C. and J.-S.Y. performed mass spectrometry analysis. S.Y. and S.M. generated tm3718. D.X. conceived and supervised the project, analysed data, and wrote the manuscript together with Y.P., M.Z., L.Z., Q.L. and H.L.

Correspondence to Ding Xue.

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The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks A. Dillin and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Conditioned medium generated from UV or IR and purified tCPR-4 proteins cause embryonic lethality.

a, The embryonic lethality rate of wild-type (N2) or cep-1(gk138) animals after 100 J m−2 UV irradiation or 500 Gy IR compared with sham-irradiation controls. b, N2 animals were used to generate UV-CM, UV-ctrl, IR-CM and IR-ctrl, which were used to treat unexposed N2 animals in the embryonic lethality assays (Methods). c, Recombinant tCPR-4 (wild type or mutant; 2.8 μM), recombinant human cathepsin B (rhCTSB; 0.27 μM) or the buffer control were used to treat N2 animals in the embryonic lethality assays. Total numbers of embryos scored: 1,781, 805, 1,249, 2,645, 596 and 1,862 embryos, from the left to the right in a; 2,721, 2,484, 880 and 743, from left to right in b; and 979, 875, 929, 939, 907 and 777, from left to right in c. Six independent assays (a, UV-ctrl and UV-CM in b) and three independent assays (IR-ctrl and IR-CM in b, c) were performed for each condition. Data are mean ± s.e.m. NS, not significant; **P < 0.01, ***P < 0.001, two-sided, unpaired t-test. Source data

Extended Data Figure 2 Characterization of the nature and the source of the RIBE factors.

a, b, Treatment of UV-CM and UV-ctrl collected from N2 animals irradiated at 100 J m−2 with RNase (1 μg μl−1) or DNase (0.01 U μl−1) did not alter the apoptosis-inhibitory effect on ced-1(e1735) animals (Methods). Germ-cell corpses were scored after 48-h treatment of ced-1(e1735) L4 larvae. c, eg, ced-1(e1735) L4 larvae were treated with UV-CM and UV-ctrl (0.1 μg μl−1) prepared from ced-3(n2433) animals (c), glp-1(e2141ts) animals grown at 25 °C (e), N2 animals fed with formaldehyde-treated HB101 bacteria (f), and Pcpr-4::cpr-4::flag; cpr-4(tm3718) animals with or without anti-Flag depletion (g), respectively. Data are mean ± s.e.m. The numbers of gonad arms scored are indicated inside the bars (ac, eg). **P < 0.01, ***P < 0.001, two-sided, unpaired t-test. d, Representative differential interference contrast (DIC) images (at least 10) of N2 and glp-1(e2141) adult animals grown at 25 °C. The gonads of the N2 animal with multiple oocytes and fertilized eggs are outlined with dash lines. glp-1(e2141) animal had no visible germ line. Scale bars, 100 μm. h, Immunoblotting analysis of secreted CPR-4::Flag in UV-CM and UV-ctrl prepared from Pcpr-4::cpr-4::flag; cpr-4(tm3718) animals with or without anti-Flag depletion treatment. For gel source data, see Supplementary Fig. 1. Source data

Extended Data Figure 3 Identification of CPR-4 as the RIBE factor.

a, Full medium, the >10 kDa fraction, and the <10 kDa fraction of UV-CM and UV-ctrl derived from N2 animals were used to treat ced-1(e1735) animals in germ-cell corpse assays as in Fig. 1b. Data are mean ± s.e.m. The numbers of gonad arms scored are indicated inside the bars. b, Identification of CPR-4 as the RIBE factor through the RNAi screen. UV-ctrl and UV-CM prepared from RNAi-treated animals were used to treat ced-1(e1735) animals. The number of germ-cell corpse decrease (y-axis) was calculated by subtracting the number of average germ-cell corpses under UV-ctrl treatment from that under UV-CM treatment. Among the candidate genes, RNAi of eft-3, ubq-2 and act-1 caused strong embryonic lethality and we were unable to obtain their UV-CM. RNAi of his-1, his-4 and his-71 caused partial embryonic lethality. 20 gonad arms were scored in each RNAi experiment. c, Secretion of CPR-4::Flag into UV-CM was greatly reduced in irradiated cep-1(gk138) animals carrying a single-copy integration of Pcpr-4::cpr-4::flag compared with that from irradiated N2 animals carrying the same Pcpr-4::cpr-4::flag transgene. Concentrated UV-CM or UV-ctrl (1 μg μl−1) from the indicated strains was subjected to the immunoblotting analysis using an antibody to the Flag epitope. d, The protease activity of 0.27 μM rhCTSB or 2.8 μM recombinant tCPR-4 protein was measured as in Fig. 2b. Data are mean ± s.e.m. (n = 6 in each assay). e, rhCTSB (0.27 μM) or the buffer control was used to treat ced-1(e1735) animals. Animals cultured in the rhCTSB buffer grew slower than in the tCPR-4 buffer and had less germ-cell corpses. Data are mean ± s.e.m. (n = 21 in each assay). Germ-cell corpses were scored after 48 h treatment (a, b, e). ***P < 0.001, two-sided, unpaired t-test. For gel source data, see Supplementary Fig. 1. Source data

Extended Data Figure 4 Representative MS/MS spectra from LTQ-Orbitrap used to confirm the identity of CPR-4 in UV-CM.

a, Tryptic peptides of protein band 6 in the SDS–PAGE gel (Fig. 1d) were analysed by LC–MS/MS using LTQ-Orbitrap. The amino acid sequences of peptides identified by MS/MS analysis and matched to the amino acid sequences of CPR-4 are underlined and in red. b, The MS/MS spectra of the two peptides identified in a are shown. The assignments of the fragmented ions observed to specific amino acid residues were performed using the Scaffold 3 search engine, and the search results are shown below the MS/MS spectra. The lower-case ‘c’ indicates the carbamidomethyl-modified cysteine residue in the tryptic peptide.

Extended Data Figure 5 The cpr-4 deletion mutation and sequence alignment of human and mouse cathepsin B and CPR-4.

a, A schematic representation of the cpr-4 gene structure and the tm3718 deletion. Exons are depicted as blue boxes and introns and the untranslated region as lines. The red box indicates the region of cpr-4 removed by the 406-bp tm3718 deletion. The green box indicates a 12-bp insertion. b, Sequence alignment of human cathepsin B, mouse cathepsin B and CPR-4. Residues that are identical in all three proteins are shaded in pink. The two catalytic residues are shaded in green, which are the active-site cysteine residue that serves as a nucleophile and the histidine residue that acts as a general base to facilitate hydrolysis of the peptide bonds of the substrates16, respectively.

Extended Data Figure 6 Analysis of the roles of additional genes in mediating RIBEs.

a, LUI assays. Animals of the indicated genotype were analysed for the bystander Phsp-4::gfp response 24 h after localized irradiation at the head region as described in Fig. 3. Data are mean ± s.e.m. The numbers of animals scored are indicated inside the bars. b, Germ-cell corpse assays after tCPR-4 treatment. 2.8 μM recombinant tCPR-4 protein or buffer control was used to treat L4 larvae of the indicated genotype as described in Fig. 4a. Data are mean ± s.e.m. The numbers of gonad arms scored are indicated inside the bars. c, Immunoblotting analysis of secreted CPR-4::Flag in UV-CM and UV-ctrl from Pcpr-4::cpr-4::flag; daf-2(e1370); cpr-4(tm3718) animals was done as in Fig. 1f. d, Germ-cell corpse assays. ced-1(e1735) L4 larvae were treated with UV-CM and UV-ctrl (0.1 μg μl−1) prepared from c. Data are mean ± s.e.m. The numbers of gonad arms scored are indicated inside the bars. e, Germ-cell proliferation assays. N2 and cep-1(gk138) L4 larvae were treated in S-medium containing 2.8 μM recombinant tCPR-4 or buffer control for 48 h. The numbers of nuclei and metaphase nuclei in the mitotic zone of the germ line were scored (Methods). Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, two-sided, unpaired t-test. For gel source data, see Supplementary Fig. 1. Source data

Extended Data Figure 7 The expression patterns of cpr-4 in C. elegans.

ag, i, Representative GFP and DIC images (at least 15 each) of N2 animals (ag) or cep-1(gk138) animals (i) carrying a single-copy integration of Pcpr-4::nls::gfp at the indicated developmental stages. Arrows point to the embryo and the L1 larva that showed no or very dim GFP (a, b). Scale bar, 100 μm. h, Representative DIC, GFP and DIC and GFP merged images (at least 15) of a L4 larva carrying the same Pcpr-4::nls::gfp transgene (left), and corresponding tenfold magnified images showing GFP expression in intestinal cells (right). GFP was seen mostly in the nuclei (arrows). Scale bars, 100 μm (left) and 10 μm (right). j, The intensity of GFP fluorescence in Pcpr-4::nls::gfp and cep-1(gk138); Pcpr-4::nls::gfp animals at different developmental stages was quantified using the Image J software. Data are mean ± s.e.m. n = 28, 28, 24, 31, 30, 33, 52, 52, 19, 28, 52, 52, 24 and 25 animals, scored from left to right, respectively. **P < 0.01, ***P < 0.001, two-sided, unpaired t-test. k, Quantification of GFP intensity in N2 and cep-1(gk138) animals carrying the same single-copy Pcpr-4::nls::gfp transgene irradiated by UV or sham-irradiated, using Image J. Data are mean ± s.e.m. n = 38, 37, 32 and 30 animals, scored from left to right, respectively. ***P < 0.001, two-sided, unpaired t-test. Source data

Extended Data Figure 8 Pharyngeal expression of CPR-4 results in embryonic lethality, larval arrest and reduced germ-cell death.

a, b, Representative DIC and mCherry images (at least 10) of adult animals with pharyngeal expression of CPR-4::mCherry (a) and tCPR-4::mCherry (b). White dashed lines highlight the edge of the pharynx. Arrowheads indicate cells, including coelomocytes, that had taken up CPR-4::mCherry (a), which was made in and secreted from the pharynx and transported to other parts of the animal, probably through the pseudocoelom, a fluid-filled body cavity. The enlarged images of two pairs of posterior cells with weak fluorescence (indicated by colour arrowheads) are shown in dashed boxes with corresponding colours. Scale bars, 100 μm. c, The percentages of embryonic lethality and larval arrest were scored in embryos or larvae carrying Pmyo-2::CPR-4::mCherry (wild-type or mutant) or Pmyo-2::tCPR-4::mCherry transgenes. Three independent transgenic lines were scored for each construct. The number of newly hatched transgenic L1 larvae scored and the number of transgenic embryos scored are indicated in parentheses. The increased larval arrest seen in Pmyo-2::CPR-4::mCherry transgenic animals was blocked when transgenic animals were treated with cpr-4 RNAi (Extended Data Table 2), indicating that reducing cpr-4 expression prevents larval arrest. All animals carry the ced-1(e1735) and cpr-4(tm3718) mutations (ac). d, Quantification of germ-cell corpses in transgenic animals. L4 ced-1(e1735); cpr-4(tm3718) animals carrying the indicated transgenes were grown on regular NGM plates for 24 h before examination. Data are mean ± s.e.m. The numbers of gonad arms scored are indicated inside the bars. ***P < 0.001, one-way analysis of variance (ANOVA). Source data

Extended Data Table 1 A summary of peptide identification information in bands 1–10 by LC–MS/MS analysis using LTQ-Orbitrap
Extended Data Table 2 cpr-4 RNAi treatment of Pmyo-2::CPR-4::mCherry transgenic animals

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Peng, Y., Zhang, M., Zheng, L. et al. Cysteine protease cathepsin B mediates radiation-induced bystander effects. Nature 547, 458–462 (2017) doi:10.1038/nature23284

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