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Cardiac glycosides are potent inhibitors of interferon-β gene expression

Abstract

Here we report that bufalin and other cardiac glycoside inhibitors of the sodium-potassium ATPase (sodium pump) potently inhibit the induction of the interferon-β (IFNβ) gene by virus, double-stranded RNA or double-stranded DNA. Cardiac glycosides increase the intracellular sodium concentration, which appears to inhibit the ATPase activity of the RNA sensor RIG-I, an essential and early component in the IFNβ activation pathway. This, in turn, prevents the activation of the critical transcription factors IRF3 and NFκB. Bufalin inhibition can be overcome by expressing a drug-resistant variant of the sodium pump and knocking down the pump by short hairpin RNA inhibits IFNβ expression. Thus, bufalin acts exclusively through the sodium pump. We also show that bufalin inhibits tumor necrosis factor (TNF) signaling, at least in part by interfering with the nuclear translocation of NFκB. These findings suggest that bufalin could be used to treat inflammatory and autoimmune diseases in which IFN or TNF are hyperactivated.

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Figure 1: Bufalin potently blocks virus-, dsRNA- and dsDNA-induced gene expression.
Figure 2: Bufalin inhibits virus-induced IRF3 and p65 activation.
Figure 3: RIG-I ATPase activity is inhibited by bufalin treatment.
Figure 4: Bufalin inhibits IFNβ induction exclusively through the sodium pump.
Figure 5: Knocking down sodium pump expression inhibits IFNβ induction.
Figure 6: Bufalin inhibits TNF signaling.

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References

  1. Sen, G.C. Viruses and interferons. Annu. Rev. Microbiol. 55, 255–281 (2001).

    Article  CAS  Google Scholar 

  2. Honda, K., Takaoka, A. & Taniguchi, T. Type I interferon [corrected] gene induction by the interferon regulatory factor family of transcription factors. Immunity 25, 349–360 (2006).

    Article  CAS  Google Scholar 

  3. Chiu, Y.H., Macmillan, J.B. & Chen, Z.J. RNA polymerase III detects cytosolic DNA and induces type I interferons through the RIG-I pathway. Cell 138, 576–591 (2009).

    Article  CAS  Google Scholar 

  4. Ablasser, A. et al. RIG-I-dependent sensing of poly(dA:dT) through the induction of an RNA polymerase III-transcribed RNA intermediate. Nat. Immunol. 10, 1065–1072 (2009).

    Article  CAS  Google Scholar 

  5. García-Sastre, A. & Biron, C.A. Type 1 interferons and the virus-host relationship: a lesson in detente. Science 312, 879–882 (2006).

    Article  Google Scholar 

  6. Le Bon, A. & Tough, D.F. Links between innate and adaptive immunity via type I interferon. Curr. Opin. Immunol. 14, 432–436 (2002).

    Article  CAS  Google Scholar 

  7. Banchereau, J. & Pascual, V. Type I interferon in systemic lupus erythematosus and other autoimmune diseases. Immunity 25, 383–392 (2006).

    Article  CAS  Google Scholar 

  8. Hall, J.C. & Rosen, A. Type I interferons: crucial participants in disease amplification in autoimmunity. Nat. Rev. Rheumatol. 6, 40–49 (2010).

    Article  CAS  Google Scholar 

  9. Mandl, J.N. et al. Divergent TLR7 and TLR9 signaling and type I interferon production distinguish pathogenic and nonpathogenic AIDS virus infections. Nat. Med. 14, 1077–1087 (2008).

    Article  CAS  Google Scholar 

  10. Whittemore, L.A. & Maniatis, T. Postinduction turnoff of beta-interferon gene expression. Mol. Cell. Biol. 10, 1329–1337 (1990).

    Article  CAS  Google Scholar 

  11. Jacquelin, B. et al. Nonpathogenic SIV infection of African green monkeys induces a strong but rapidly controlled type I IFN response. J. Clin. Invest. 119, 3544–3555 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Maniatis, T. et al. Structure and function of the interferon-beta enhanceosome. Cold Spring Harb. Symp. Quant. Biol. 63, 609–620 (1998).

    Article  CAS  Google Scholar 

  13. Sun, L., Liu, S. & Chen, Z.J. SnapShot: pathways of antiviral innate immunity. Cell 140, 436–436.e2 (2010).

    Article  Google Scholar 

  14. Ford, E. & Thanos, D. The transcriptional code of human IFN-beta gene expression. Biochim. Biophys. Acta 1799, 328–336 (2010).

    Article  CAS  Google Scholar 

  15. Kato, H. et al. Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature 441, 101–105 (2006).

    Article  CAS  Google Scholar 

  16. Cui, S. et al. The C-terminal regulatory domain is the RNA 5′-triphosphate sensor of RIG-I. Mol. Cell 29, 169–179 (2008).

    Article  CAS  Google Scholar 

  17. Takahasi, K. et al. Nonself RNA-sensing mechanism of RIG-I helicase and activation of antiviral immune responses. Mol. Cell 29, 428–440 (2008).

    Article  CAS  Google Scholar 

  18. Seth, R.B., Sun, L., Ea, C.K. & Chen, Z.J. Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3. Cell 122, 669–682 (2005).

    Article  CAS  Google Scholar 

  19. Tang, E.D. & Wang, C.Y. MAVS self-association mediates antiviral innate immune signaling. J. Virol. 83, 3420–3428 (2009).

    Article  CAS  Google Scholar 

  20. Fujita, T. A nonself RNA pattern: tri-p to panhandle. Immunity 31, 4–5 (2009).

    Article  CAS  Google Scholar 

  21. Myong, S. et al. Cytosolic viral sensor RIG-I is a 5′-triphosphate-dependent translocase on double-stranded RNA. Science 323, 1070–1074 (2009).

    Article  CAS  Google Scholar 

  22. Gack, M.U. et al. TRIM25 RING-finger E3 ubiquitin ligase is essential for RIG-I-mediated antiviral activity. Nature 446, 916–920 (2007).

    Article  CAS  Google Scholar 

  23. Gack, M.U., Nistal-Villan, E., Inn, K.S., Garcia-Sastre, A. & Jung, J.U. Phosphorylation-mediated negative regulation of RIG-I antiviral activity. J. Virol. 84, 3220–3229 (2010).

    Article  CAS  Google Scholar 

  24. Saha, S.K. et al. Regulation of antiviral responses by a direct and specific interaction between TRAF3 and Cardif. EMBO J. 25, 3257–3263 (2006).

    Article  CAS  Google Scholar 

  25. Guo, B. & Cheng, G. Modulation of the interferon antiviral response by the TBK1/IKKi adaptor protein TANK. J. Biol. Chem. 282, 11817–11826 (2007).

    Article  CAS  Google Scholar 

  26. Kawai, T. et al. IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction. Nat. Immunol. 6, 981–988 (2005).

    Article  CAS  Google Scholar 

  27. Ishikawa, H. & Barber, G.N. STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling. Nature 455, 674–678 (2008).

    Article  CAS  Google Scholar 

  28. Zhong, B. et al. The adaptor protein MITA links virus-sensing receptors to IRF3 transcription factor activation. Immunity 29, 538–550 (2008).

    Article  CAS  Google Scholar 

  29. Schröder, M., Baran, M. & Bowie, A.G. Viral targeting of DEAD box protein 3 reveals its role in TBK1/IKKepsilon-mediated IRF activation. EMBO J. 27, 2147–2157 (2008).

    Article  Google Scholar 

  30. Prassas, I. & Diamandis, E.P. Novel therapeutic applications of cardiac glycosides. Nat. Rev. Drug Discov. 7, 926–935 (2008).

    Article  CAS  Google Scholar 

  31. Hemmi, H. et al. The roles of two IkappaB kinase-related kinases in lipopolysaccharide and double stranded RNA signaling and viral infection. J. Exp. Med. 199, 1641–1650 (2004).

    Article  CAS  Google Scholar 

  32. Fitzgerald, K.A. et al. IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway. Nat. Immunol. 4, 491–496 (2003).

    Article  CAS  Google Scholar 

  33. Yoneyama, M. et al. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat. Immunol. 5, 730–737 (2004).

    Article  CAS  Google Scholar 

  34. Saito, T. et al. Regulation of innate antiviral defenses through a shared repressor domain in RIG-I and LGP2. Proc. Natl. Acad. Sci. USA 104, 582–587 (2007).

    Article  CAS  Google Scholar 

  35. Langer, G.A. Ionic basis of myocardial contractility. Annu. Rev. Med. 28, 13–20 (1977).

    Article  CAS  Google Scholar 

  36. Lingrel, J.B. The physiological significance of the cardiotonic steroid/ouabain-binding site of the Na,K-ATPase. Annu. Rev. Physiol. 72, 395–412 (2010).

    Article  CAS  Google Scholar 

  37. Ohtsubo, M., Noguchi, S., Takeda, K., Morohashi, M. & Kawamura, M. Site-directed mutagenesis of Asp-376, the catalytic phosphorylation site, and Lys-507, the putative ATP-binding site, of the alpha-subunit of Torpedo californica Na+/K(+)-ATPase. Biochim. Biophys. Acta 1021, 157–160 (1990).

    Article  CAS  Google Scholar 

  38. Simpson, C.D. et al. Inhibition of the sodium potassium adenosine triphosphatase pump sensitizes cancer cells to anoikis and prevents distant tumor formation. Cancer Res. 69, 2739–2747 (2009).

    Article  CAS  Google Scholar 

  39. Izquierdo, I. Nimodipine and the recovery of memory. Trends Pharmacol. Sci. 11, 309–310 (1990).

    Article  CAS  Google Scholar 

  40. Trube, G., Rorsman, P. & Ohno-Shosaku, T. Opposite effects of tolbutamide and diazoxide on the ATP-dependent K+ channel in mouse pancreatic beta-cells. Pflugers Arch. 407, 493–499 (1986).

    Article  CAS  Google Scholar 

  41. Garvin, J.L., Simon, S.A., Cragoe, E.J. Jr. & Mandel, L.J. Phenamil: an irreversible inhibitor of sodium channels in the toad urinary bladder. J. Membr. Biol. 87, 45–54 (1985).

    Article  CAS  Google Scholar 

  42. Xie, Z. & Cai, T. Na+-K+–ATPase-mediated signal transduction: from protein interaction to cellular function. Mol. Interv. 3, 157–168 (2003).

    Article  CAS  Google Scholar 

  43. Gee, P. et al. Essential role of the N-terminal domain in the regulation of RIG-I ATPase activity. J. Biol. Chem. 283, 9488–9496 (2008).

    Article  CAS  Google Scholar 

  44. Cyert, M.S. Regulation of nuclear localization during signaling. J. Biol. Chem. 276, 20805–20808 (2001).

    Article  CAS  Google Scholar 

  45. Yang, Q. et al. Cardiac glycosides inhibit TNF-alpha/NF-kappaB signaling by blocking recruitment of TNF receptor-associated death domain to the TNF receptor. Proc. Natl. Acad. Sci. USA 102, 9631–9636 (2005).

    Article  CAS  Google Scholar 

  46. Ronnblom, L. & Elkon, K.B. Cytokines as therapeutic targets in SLE. Nat. Rev. Rheumatol. 6, 339–347 (2010).

    Article  Google Scholar 

  47. Meng, Z. et al. Pilot study of huachansu in patients with hepatocellular carcinoma, nonsmall-cell lung cancer, or pancreatic cancer. Cancer 115, 5309–5318 (2009).

    Article  Google Scholar 

  48. Chen, S. et al. A small molecule that directs differentiation of human ESCs into the pancreatic lineage. Nat. Chem. Biol. 5, 258–265 (2009).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work is supported by US National Institutes of Health grant 5R01AI020642-26 (to T.M.). S.C. is supported by the postdoctoral fellowship from Juvenile Diabetes Research Foundation. We thank X. Sun (Brandeis University) for help with the microarray analysis. We also thank S.-L. Ng for critical reading of this manuscript and S. Schalm for helpful discussions.

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J.Y., S.C. and T.M. conceived the research, J.Y. and S.C. conducted experiments, and J.Y. and T.M. wrote the paper.

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Correspondence to Tom Maniatis.

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The authors have filed a patent on the inhibition of interferon-β expression by cardiac glycosides described in the paper.

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Supplementary Methods, Supplementary Table 1 and Supplementary Figures 1–12 (PDF 13254 kb)

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Ye, J., Chen, S. & Maniatis, T. Cardiac glycosides are potent inhibitors of interferon-β gene expression. Nat Chem Biol 7, 25–33 (2011). https://doi.org/10.1038/nchembio.476

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