doi: 10.3967/bes2019.025
Connexin43 Modulates X-Ray-Induced Pyroptosis in Human Umbilical Vein Endothelial Cells
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Abstract:
Objective Pyroptosis is an inflammatory form of programmed cell death. This phenomenon has been recently reported to play an important role in radiation-induced normal tissue injury. Connexin43 (Cx43) is a gap junction protein that regulates cell growth and apoptosis. In this study, we investigated the effect of Cx43 on X-ray-induced pyroptosis in the human umbilical vein endothelial cells (HUVECs). Methods HUVECs, Cx43 overexpression, and Cx43 knockdown strains were irradiated with 10 Gy. Proteins were detected using western blot analysis. Cell pyroptosis was evaluated using the fluorescence-labeled inhibitor of caspase assay (FLICA) and propidium iodide staining through flow cytometry and confocal microscopy. Cell morphology and cytotoxicity were detected by scanning electron microscopy and lactate dehydrogenase release assay, respectively. Results Irradiation with 10 Gy X-ray induced pyroptosis in the HUVECs and reduced Cx43 expression. The pyroptosis in the HUVECs was significantly attenuated by overexpression of Cx43 as it decreased the level of active caspase-1. However, interference of Cx43 expression with siRNA significantly promoted pyroptosis by increasing the active caspase-1 level. Pannexin1 (Panx1), a gap junction protein regulates pyroptosis, and its cleaved form is used to evaluate channel opening and active state. The level of cleaved Panx1 in the HUVECs and Cx43 knockdown strains increased in the presence of X-ray, but decreased in the Cx43 overexpression strains. Furthermore, interference of Panx1 with siRNA alleviated the upregulation of pyroptosis caused by Cx43 knockdown. Conclusion Results suggest that single high-dose X-ray irradiation induces pyroptosis in the HUVECs. In addition, Cx43 regulates pyroptosis directly by activating caspase-1 or indirectly by cleaving Panx1. -
Key words:
- X-ray /
- Connexin43 /
- HUVECs /
- Pyroptosis
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Figure 1. X-ray induces pyroptosis in the HUVECs. (A) Cleaved caspase-1 (P20) was detected after 6 h up to 48 h in the HUVECs after being irradiated with 10 Gy X-ray. (B) Cleaved caspase-1 (P20) was detected in groups subjected to different X-ray doses (2.5, 5, 10, and 20 Gy) at 12 h after irradiation. (C) HUVECs with active caspase-1 (those stained with FLICA appear green) were determined using by confocal microscopy. (D) Percentage of pyroptotic HUVECs was determined with active caspase-1 (those stained with FLICA are represented in the abscissa) and that of PI (represent in ordinate) was determined via flow cytometry, and statistical analyses of the percentage of PI+-active caspase-1+ between control (con) and group irradiated with 10 Gy after 72 h are on the right panel. (E) The morphology of the HUVECs was detected by transmission electron microscopy in the control and 10 Gy groups. (F) LDH release was examined at 0-72 h after irradiation and (G) with different doses (2.5, 5, 10, and 20 Gy) at 72 h after irradiation. The results are presented as means ± SEM. Data were obtained from the three independent experiments. Symbols *, **, and *** represent P < 0.05, P < 0.01, and P < 0.001, respectively. Scale bar = 10 μm.
Figure 2. Exposure to X-ray reduces expression of Cx43. (A) Cx43 expression after irradiation with 10 Gy at 0-72 h was detected using western blot analysis. (B) Expression of Cx43 after 12 h of 2.5-10 Gy irradiation as detected by western blot analysis. The results are presented as means ± SEM. Data were obtained from three independent experiments. Symbols *, **, and *** represent P < 0.05, P < 0.01, and P < 0.001, respectively.
Figure 3. Overexpression of Cx43 decreased pyroptosis in the X-ray-irradiated HUVECs. All the experiments were carried out in the control, irradiation (IR), irradiation with vector (IR-pCMV5), and irradiation with Cx43 overexpression (IR-pCMV-Cx43) groups. (A) Percentage of pyroptotic HUVECs with active caspase-1 (stained with FLICA represented in abscissa) and PI (represented in ordinate) was determined via flow cytometry, and statistical analysis of the percentage of PI+-active caspase-1+ and active caspase-1+ HUVECs between the groups after 72 h is presented in the right panel. (B) HUVECs with active caspase-1 (stained with FLICA appear green) were determined using the confocal microscope. (C) Cleaved caspase-1 (P20) and Cx43 were detected at 12 h in HUVECs after irradiation with 10 Gy. (D) The LDH released in the supernatant of each group was examined. The results are presented as means ± SEM. Data was obtained from three independent experiments. Symbols ** and *** represent P < 0.01 and P < 0.001, vs. IR-pCMV5, respectively. Scale bar = 10 μm.
Figure 4. Cx43 knockdown increases pyroptosis in the X-ray-irradiated HUVECs. All the experiments were carried out in the control, irradiation (IR), irradiation with scramble (IR-scr), and irradiation with Cx43 knockdown (IR-siCx43) groups. (A) Percentage of pyroptotic HUVECs was determined with active caspase-1 (stained with FLICA represent in abscissa) and PI (represent in ordinate) via flow cytometry, and statistical analysis of the percentage of PI+-active caspase-1+ and active caspase-1+ HUVECs between groups after 72 h is presented in the right panel. (B) HUVECs with active caspase-1 (stained with FLICA appear green) were determined by confocal microscopy. (C) Cleaved caspase-1 (P20) and Cx43 were detected at 12 h in HUVECs after irradiation with 10 Gy. (D) LDH release from the supernatant of each group. (E) Morphology of HUVECs transfected with vector scr or siCx43 irradiated with 10 Gy after 72 h, as detected by transmission electron microscopy. The results are presented as means ± SEM. Data were obtained from three independent experiments. Symbols * and ** represent P < 0.05 and P < 0.01, vs. IR-scr, respectively. Scale bar = 10 μm.
Figure 5. Cx43 affects Pannexin-1 in X-ray-irradiated HUVECs. (A) Cleaved Pannexin-1 was detected between 0-72 h in HUVECs after irradiated by 10 Gy X-ray. (B) Cleaved Pannexin-1 expression level in HUVECs after 0, 2.5, 5, 10, and 20 Gy radiation at 12 h. (C) Cleaved Pannexin-1 and Cx43 expression in HUVECs after 10 Gy radiation at 12 h in control, irradiation (IR), irradiation with vector (IR-pCMV5), and irradiation with Cx43 overexpression (IR-pCMV-Cx43) groups. (D) Cleaved Pannexin-1 and Cx43 expression in HUVECs after 10 Gy radiation at 12 h in control, irradiation (IR), irradiation with scramble (IR-scr), and irradiation with Cx43 knockdown (IR-siCx43) groups. The results are presented as means ± SEM. Data were obtained from three independent experiments. Symbols *, **, and *** represent P < 0.05, P < 0.01, and P < 0.001, respectively.
Figure 6. Cx43 mediates pyroptosis through cleaved Pannexin1. All the experiments were carried out in the control, irradiation (IR), irradiation with scramble (IR-scr), irradiation with Panx1 knockdown (IR-siPanx1), irradiation with Cx43 knockdown (IR-siCx43), and irradiation with both Cx43 knockdown and Panx1 knockdown (IR-siCx43-siPanx1). (A) Percentage of pyroptotic HUVECs was determined with active caspase-1 (stained with FLICA represent in abscissa) and PI (represent in ordinate) via flow cytometry, and statistical analysis of percentage of PI+-active caspase-1+ and active caspase-1+ HUVECs between groups after 72 h is presented in the right panel. (B) HUVECs with active caspase-1 (stained with FLICA appear green) were determined via confocal microscopy. (C) Cleaved caspase-1 (P20) was detected using western-blot analysis at 12 h after 10 Gy X-ray irradiation in each group. (D) LDH release from the supernatant of each group was examined by the LDH release essay. The results are presented as means ± SEM. Data was obtained from three independent experiments. Symbols *, **, and *** represent P < 0.05, P < 0.01, and P < 0.001 respectively. Scale bar = 10 μm.
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[1] Formenti SC, Lymberis SC, Dewyngaert JK. Ischemic heart disease after breast cancer radiotherapy. N Engl J Med, 2013; 368, 2525. http://d.old.wanfangdata.com.cn/Periodical/zgyszz201310014 [2] Shimizu Y, Kodama K, Nishi N, et al. Radiation exposure and circulatory disease risk: Hiroshima and Nagasaki atomic bomb survivor data, 1950-2003. BMJ, 2010; 340, b5349. doi: 10.1136/bmj.b5349 [3] Korpela E, Liu SK. Endothelial perturbations and therapeutic strategies in normal tissue radiation damage. Radiat Oncol, 2014; 9, 1-9. doi: 10.1186/1748-717X-9-1 [4] Corre I, Guillonneau M, Paris F. Membrane signaling induced by high doses of ionizing radiation in the endothelial compartment. Relevance in radiation toxicity. Int J Mol Sci, 2013; 14, 22678-96. doi: 10.3390/ijms141122678 [5] Venkatesulu BP, Mahadevan LS, Aliru ML, et al. Radiation-Induced Endothelial Vascular Injury: A Review of Possible Mechanisms. JACC Basic Transl Sci, 2018; 3, 563-72. doi: 10.1016/j.jacbts.2018.01.014 [6] Liu YG, Chen JK, Zhang ZT, et al. NLRP3 inflammasome activation mediates radiation-induced pyroptosis in bone marrow-derived macrophages. Cell Death Dis, 2017; 8, e2579. doi: 10.1038/cddis.2016.460 [7] Liao H, Wang H, Rong X, et al. Mesenchymal stem cells attenuate radiation-induced brain injury by inhibiting microglia pyroptosis. Biomed Res Int, 2017; 2017, 1948985. http://europepmc.org/articles/PMC6020670/ [8] Bergsbaken T, Fink SL, Cookson BT. Pyroptosis: host cell death and inflammation. Nat Rev Microbiol, 2009; 7, 99-109. doi: 10.1038/nrmicro2070 [9] Shi J, Gao W, Shao F. Pyroptosis: Gasdermin-mediated programmed necrotic cell death. Trends Biochem Sci, 2017; 42, 245-54. doi: 10.1016/j.tibs.2016.10.004 [10] Xi H, Zhang Y, Xu Y, et al. Caspase-1 inflammasome activation mediates homocysteine-induced pyrop-apoptosis in endothelial cells. Circ Res, 2016; 118, 1525-39. doi: 10.1161/CIRCRESAHA.116.308501 [11] Abe J, Morrell C. Pyroptosis as a regulated form of necrosis: PI+/Annexin V-/High Caspase 1/Low Caspase 9 activity in cells = pyroptosis? Circ Res, 2016; 118, 1457-60. doi: 10.1161/CIRCRESAHA.116.308699 [12] Miao EA, Leaf IA, Treuting PM, et al. Caspase-1-induced pyroptosis is an innate immune effector mechanism against intracellular bacteria. Nat Immunol, 2010; 11, 1136-42. doi: 10.1038/ni.1960 [13] Lamkanfi M, Dixit VM. Mechanisms and functions of inflammasomes. Cell, 2014; 157, 1013-22. doi: 10.1016/j.cell.2014.04.007 [14] Schroder K, Tschopp J. The inflammasomes. Cell, 2010; 140, 821-32. doi: 10.1016/j.cell.2010.01.040 [15] Franklin BS, Bossaller L, De Nardo D, et al. The adaptor ASC has extracellular and ['p]ionoid' activities that propagate inflammation. Nat Immunol, 2014; 15, 727-37. doi: 10.1038/ni.2913 [16] Baroja-Mazo A, Martín-Sánchez F, Gomez AI, et al. The NLRP3 inflammasome is released as a particulate danger signal that amplifies the inflammatory response. Nat Immunol, 2014; 15, 738. doi: 10.1038/ni.2919 [17] Skerrett IM, Williams JB. A structural and functional comparison of gap junction channels composed of connexins and innexins. Dev Neurobiol, 2017; 77, 522-47. doi: 10.1002/dneu.v77.5 [18] Dempsie Y, Martin P, Upton PD. Connexin-mediated regulation of the pulmonary vasculature. Biochem Soc Trans, 2015; 43, 524-9. doi: 10.1042/BST20150030 [19] Zhang J, O'Carroll SJ, Henare K, et al. Connexin hemichannel induced vascular leak suggests a new paradigm for cancer therapy. FEBS Lett, 2014; 588, 1365-71. doi: 10.1016/j.febslet.2014.02.003 [20] De Bock M, Wang N, Decrock E, et al. Intracellular cleavage of the Cx43 c-terminal domain by matrix-metalloproteases: A novel contributor to inflammation? Mediat Inflamm, 2015; 2015, 1-18. http://europepmc.org/abstract/MED/26424967 [21] Taylor KA, Wright JR, Mahaut-Smith MP. Regulation of pannexin-1 channel activity. Biochem Soc Trans, 2015; 43, 502-7. doi: 10.1042/BST20150042 [22] Thi MM, Islam S, Suadicani SO, et al. Connexin43 and pannexin-1 channels in osteoblasts: who is the "hemichannel"? J Membr Biol, 2012; 245, 401-9. doi: 10.1007/s00232-012-9462-2 [23] Li S, Tomic M, Stojilkovic SS. Characterization of novel pannexin 1 isoforms from rat pituitary cells and their association with ATP-gated P2X channels. Gen Comp Endocrinol, 2011; 174, 202-10. doi: 10.1016/j.ygcen.2011.08.019 [24] Pelegrin P, Surprenant A. Pannexin-1 mediates large pore formation and interleukin-1beta release by the ATP-gated P2X7 receptor. EMBO J, 2006; 25, 5071-82. doi: 10.1038/sj.emboj.7601378 [25] Rafii S, Ginsberg M, Scandura J, et al. Transplantation of Endothelial Cells to Mitigate Acute and Chronic Radiation Injury to Vital Organs. Radiat Res, 2016; 186, 196-202. doi: 10.1667/RR14461.1 [26] Slezak J, Kura B, Ravingerova T, et al. Mechanisms of cardiac radiation injury and potential preventive approaches. Can J Physiol Pharmacol, 2015; 93, 737-53. doi: 10.1139/cjpp-2015-0006 [27] Wu D, Han R, Deng S, et al. Protective effects of flagellin A N/C against radiation-induced NLR Pyrin domain containing 3 inflammasome-dependent pyroptosis in Intestinal Cells. Int J Radiat Oncol Biol Phys, 2018; 101, 107-17. doi: 10.1016/j.ijrobp.2018.01.035 [28] Han R, Wu D, Deng S, et al. NLRP3 inflammasome induces pyroptosis in lung tissues of radiation-induced lung injury in mice. Chinese J Cel & Mol Immunol, 2017; 33, 1206-11. (In Chinese) http://d.old.wanfangdata.com.cn/Periodical/xbyfzmyxzz201709011 [29] Pecoraro M, Pinto A, Popolo A. Inhibition of connexin 43 translocation on mitochondria accelerates CoCl2-induced apoptotic response in a chemical model of hypoxia. Toxicol In Vitro, 2018; 47, 120-8. doi: 10.1016/j.tiv.2017.11.004 [30] Du ZJ, Cui GQ, Zhang J, et al. Inhibition of gap junction intercellular communication is involved in silica nanoparticles-induced H9c2 cardiomyocytes apoptosis via the mitochondrial pathway. Int J Nanomedicine, 2017; 12, 2179-88. doi: 10.2147/IJN [31] Ghosh S, Kumar A, Chandna S. Connexin-43 downregulation in G2/M phase enriched tumour cells causes extensive low-dose hyper-radiosensitivity (HRS) associated with mitochondrial apoptotic events. Cancer lett, 2015; 363, 46-59. doi: 10.1016/j.canlet.2015.03.046 [32] Mathur A, Kumar A, Babu B, et al. In vitro mesenchymal-epithelial transition in NIH3T3 fibroblasts results in onset of low-dose radiation hypersensitivity coupled with attenuated connexin-43 response. Biochim Biophys Acta Gen Sub, 2018; 1862, 414-26. doi: 10.1016/j.bbagen.2017.11.013 [33] Autsavapromporn N, de Toledo SM, Little JB, et al. The role of gap junction communication and oxidative stress in the propagation of toxic effects among high-dose alpha-particle- irradiated human cells. Radiat Res, 2011; 175, 347-57. doi: 10.1667/RR2372.1 [34] Azzam EI, de Toledo SM, Little JB. Expression of connexin43 is highly sensitive to ionizing radiation and other environmental stresses. Cancer Res, 2003; 63, 7128-35. http://www.ncbi.nlm.nih.gov/pubmed/14612506 [35] Autsavapromporn N, De Toledo SM, Jay-Gerin JP, et al. Human cell responses to ionizing radiation are differentially affected by the expressed connexins. J Radiat Res, 2013; 54, 251-9. doi: 10.1093/jrr/rrs099 [36] Lampe PD, Tenbroek EM, Burt BJM, et al. Phosphorylation of connexin43 on serine368 by protein kinase C regulates gap junctional communication. J Cell Biol, 2000; 149, 1503-12. doi: 10.1083/jcb.149.7.1503 [37] Decrock E, Hoorelbeke D, Ramadan R, et al. Calcium, oxidative stress and connexin channels, a harmonious orchestra directing the response to radiotherapy treatment? Biochim Biophys Acta Mol Cell Res, 2017; 1864, 1099-120. doi: 10.1016/j.bbamcr.2017.02.007 [38] Li C, Meng Q, Yu X, et al. Regulatory effect of connexin 43 on basal Ca2+ signaling in rat ventricular myocytes. PLoS One, 2012; 7, e36165. doi: 10.1371/journal.pone.0036165 [39] Lee GS, Subramanian N, Kim AI, et al. The calcium-sensing receptor regulates the NLRP3 inflammasome through Ca2+ and cAMP. Nature, 2012; 492, 123-7. doi: 10.1038/nature11588 [40] Chang YY, Kao MC, Lin JA, et al. Effects of MgSO4 on inhibiting Nod-like receptor protein 3 inflammasome involve decreasing intracellular calcium. J Surg Res, 2018; 221, 257-65. doi: 10.1016/j.jss.2017.09.005 [41] Gong T, Yang Y, Jin T, et al. Orchestration of NLRP3 Inflammasome Activation by Ion Fluxes. Trends Immunol, 2018; 39, 393-406. doi: 10.1016/j.it.2018.01.009 [42] Crespo YS, Willebrords J, Johnstone SR, et al. Pannexin1 as mediator of inflammation and cell death. Biochim Biophys Acta Mol Cell Res, 2017; 1864, 51-61. doi: 10.1016/j.bbamcr.2016.10.006 [43] Draganov D, Gopalakrishnapillai S, Chen YR, et al. Modulation of P2X4/P2X7/Pannexin-1 sensitivity to extracellular ATP via Ivermectin induces a non-apoptotic and inflammatory form of cancer cell death. Sci Rep, 2015; 5, 16222. doi: 10.1038/srep16222 [44] deGassart A, Martinon F. Pyroptosis: Caspase-11? Unlocks the gates of death. Immunity, 2015; 43, 835-7. doi: 10.1016/j.immuni.2015.10.024 [45] Qu Y, Misaghi S, Newton K, et al. Pannexin-1 is required for ATP release during apoptosis but not for inflammasome activation. J Immunol, 2011; 186, 6553-61. doi: 10.4049/jimmunol.1100478 [46] Richter K, Kiefer KP, Grzesik BA, et al. Hydrostatic pressure activates ATP-sensitive K+ channels in lung epithelium by ATP release through pannexin and connexin hemichannels. FASEB J, 2014; 28, 45-55. doi: 10.1096/fj.13-229252 [47] Tonkin RS, Bowles C, Perera CJ, et al. Attenuation of mechanical pain hypersensitivity by treatment with Peptide5, a connexin-43 mimetic peptide, involves inhibition of NLRP3 inflammasome in nerve-injured mice. Exp Neurol, 2018; 300, 1-12. doi: 10.1016/j.expneurol.2017.10.016 [48] Mugisho OO, Green CR, Kho DT, et al. The inflammasome pathway is amplified and perpetuated in an autocrine manner through connexin43 hemichannel mediated ATP release. Biochim Biophys Acta Gen Subj, 2018; 1862, 385-93. doi: 10.1016/j.bbagen.2017.11.015 [49] Lohman AW, Isakson BE. Differentiating connexin hemichannels and pannexin channels in cellular ATP release. Febs Letters, 2014; 588, 1379-88. doi: 10.1016/j.febslet.2014.02.004