Abstract
Hypoxia-inducible transcription factors (HIFs) regulate hundreds of genes involved in cellular adaptation to reduced oxygen availability. HIFs consist of an O2-labile α-subunit (primarily HIF-1α and HIF-2α) and a constitutive HIF-1β subunit. In normoxia the HIF-α subunit is hydroxylated by members of a family of prolyl-4-hydroxylase domain (PHD) proteins, PHD1-3, resulting in recognition by von Hippel-Lindau protein, ubiquitination and proteasomal degradation. In contrast, reduced oxygen availability inhibits PHD activity resulting in HIF-1α stabilisation and nuclear accumulation. Nuclear import of HIF-1α mainly depends on classical nuclear localisation signals (NLS) and involves importin α/β heterodimers. Recently, a specific inhibitor of nuclear import has been identified that inhibits importin α/β-dependent import with no effects on a range of other nuclear transport pathways involving members of the importin protein family. In this study we evaluated the physiological activity of this importin α/β-inhibitor (Ivermectin) in the hypoxia response pathway. Treatment with Ivermectin decreases binding activity of HIF-1α to the importin α/β-heterodimer. Moreover, HIF-1α nuclear localisation, nuclear HIF-1α protein levels, HIF-target gene expression, as well as HIF-transcriptional activity are reduced upon Ivermectin treatment. For the first time, we demonstrate the effect of specific importin α/β-inhibition on the hypoxic response on the molecular level.
Acknowledgments
We thank W. Jelkmann for ongoing broad support and discussing the data. The authors are grateful to S. G. Schindler, B. Rudzewski, T. Svensson, P. Rouina and Swantje Häger for excellent technical support. The authors are grateful for the financial support by the University of Lübeck (gefördert mit den Mitteln der Sektion Medizin an der Universität zu Lübeck J19-2015). The authors declare no competing financial interests.
References
Ambrus, G., Whitby, L.R., Singer, E.L., Trott, O., Choi, E., Olson, A.J., Boger, D.L., and Gerace, L. (2010). Small molecule peptidomimetic inhibitors of importin α/β mediated nuclear transport. Bioorg. Med. Chem. 18, 7611–7620.10.1016/j.bmc.2010.08.038Search in Google Scholar
Ao, Z., Danappa, J.K., Wang, B., Zheng, Y., Kung, S., Rassart, E., Depping, R., Kohler, M., Cohen, E.A., and Yao, X. (2010). Importin α3 interacts with HIV-1 integrase and contributes to HIV-1 nuclear import and replication. J. Virol. 84, 8650–8663.10.1128/JVI.00508-10Search in Google Scholar
Appelhoff, R.J., Tian, Y.M., Raval, R.R., Turley, H., Harris, A.L., Pugh, C.W., Ratcliffe, P.J., and Gleadle, J.M. (2004). Differential function of the prolyl hydroxylases PHD1, PHD2, and PHD3 in the regulation of hypoxia-inducible factor. J. Biol. Chem. 279, 38458–38465.10.1074/jbc.M406026200Search in Google Scholar
Bradford, M.M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 72, 248–254.10.1016/0003-2697(76)90527-3Search in Google Scholar
Chaccour, C.J., Kobylinski, K.C., Bassat, Q., Bousema, T., Drakeley, C., Alonso, P., and Foy, B.D. (2013). Ivermectin to reduce malaria transmission: a research agenda for a promising new tool for elimination. Malar. J. 12, 153.10.1186/1475-2875-12-153Search in Google Scholar
Chachami, G., Paraskeva, E., Mingot, J.M., Braliou, G.G., Gorlich, D., and Simos, G. (2009). Transport of hypoxia-inducible factor HIF-1α into the nucleus involves importins 4 and 7. Biochem. Biophys. Res. Commun. 390, 235–240.10.1016/j.bbrc.2009.09.093Search in Google Scholar
Crump, A. and Omura, S. (2011). Ivermectin, ‘wonder drug’ from Japan: the human use perspective. Proc. Jpn. Acad. Ser. B Phys. Biol. Sci. 87, 13–28.10.2183/pjab.87.13Search in Google Scholar
Depping, R., Steinhoff, A., Schindler, S.G., Friedrich, B., Fagerlund, R., Metzen, E., Hartmann, E., and Kohler, M. (2008). Nuclear translocation of hypoxia-inducible factors (HIFs): involvement of the classical importin α/β pathway. Biochim. Biophys. Acta 1783, 394–404.10.1016/j.bbamcr.2007.12.006Search in Google Scholar
Depping, R., Jelkmann, W., and Kosyna, F.K. (2015). Nuclear-cytoplasmatic shuttling of proteins in control of cellular oxygen sensing. J. Mol. Med. (Berl) 93, 599–608.10.1007/s00109-015-1276-0Search in Google Scholar
Fontes, M.R., Teh, T., and Kobe, B. (2000). Structural basis of recognition of monopartite and bipartite nuclear localization sequences by mammalian importin-α. J. Mol. Biol. 297, 1183–1194.10.1006/jmbi.2000.3642Search in Google Scholar
Fraser, J.E., Rawlinson, S.M., Wang, C., Jans, D.A., and Wagstaff, K.M. (2014). Investigating dengue virus nonstructural protein 5 (NS5) nuclear import. Methods Mol. Biol. 1138, 301–328.10.1007/978-1-4939-0348-1_19Search in Google Scholar
Gorlich, D. and Kutay, U. (1999). Transport between the cell nucleus and the cytoplasm. Annu. Rev. Cell Dev. Biol. 15, 607–660.10.1146/annurev.cellbio.15.1.607Search in Google Scholar
Hellwig-Burgel, T., Rutkowski, K., Metzen, E., Fandrey, J., and Jelkmann, W. (1999). Interleukin-1β and tumor necrosis factor-α stimulate DNA binding of hypoxia-inducible factor-1. Blood 94, 1561–1567.10.1182/blood.V94.5.1561Search in Google Scholar
Hintersteiner, M., Ambrus, G., Bednenko, J., Schmied, M., Knox, A.J., Meisner, N.C., Gstach, H., Seifert, J.M., Singer, E.L., Gerace, L., et al. (2010). Identification of a small molecule inhibitor of importin β mediated nuclear import by confocal on-bead screening of tagged one-bead one-compound libraries. ACS Chem. Biol. 5, 967–979.10.1021/cb100094kSearch in Google Scholar
Hogarth, C.A., Calanni, S., Jans, D.A., and Loveland, K.L. (2006). Importin α mRNAs have distinct expression profiles during spermatogenesis. Dev. Dyn. 235, 253–262.10.1002/dvdy.20569Search in Google Scholar
Holscher, M., Silter, M., Krull, S., von Ahlen, M., Hesse, A., Schwartz, P., Wielockx, B., Breier, G., Katschinski, D.M., and Zieseniss, A. (2011). Cardiomyocyte-specific prolyl-4-hydroxylase domain 2 knock out protects from acute myocardial ischemic injury. J. Biol. Chem. 286, 11185–11194.10.1074/jbc.M110.186809Search in Google Scholar
Koster, M., Lykke-Andersen, S., Elnakady, Y.A., Gerth, K., Washausen, P., Hofle, G., Sasse, F., Kjems, J., and Hauser, H. (2003). Ratjadones inhibit nuclear export by blocking CRM1/exportin 1. Exp. Cell Res. 286, 321–331.10.1016/S0014-4827(03)00100-9Search in Google Scholar
Kublun, I., Ehm, P., Brehm, M.A., and Nalaskowski, M.M. (2014). Efficacious inhibition of Importin α/β-mediated nuclear import of human inositol phosphate multikinase. Biochimie 102, 117–123.10.1016/j.biochi.2014.03.001Search in Google Scholar
Kudo, N., Matsumori, N., Taoka, H., Fujiwara, D., Schreiner, E.P., Wolff, B., Yoshida, M., and Horinouchi, S. (1999). Leptomycin B inactivates CRM1/exportin 1 by covalent modification at a cysteine residue in the central conserved region. Proc. Natl. Acad. Sci. USA 96, 9112–9117.10.1073/pnas.96.16.9112Search in Google Scholar
Lee, S.H., Wolf, P.L., Escudero, R., Deutsch, R., Jamieson, S.W., and Thistlethwaite, P.A. (2000). Early expression of angiogenesis factors in acute myocardial ischemia and infarction. N. Engl. J. Med. 342, 626–633.10.1056/NEJM200003023420904Search in Google Scholar
Lokich, E., Singh, R.K., Han, A., Romano, N., Yano, N., Kim, K., and Moore, R.G. (2014). HE4 expression is associated with hormonal elements and mediated by importin-dependent nuclear translocation. Sci. Rep. 4, 5500.10.1038/srep05500Search in Google Scholar
Lundberg, L., Pinkham, C., Baer, A., Amaya, M., Narayanan, A., Wagstaff, K.M., Jans, D.A., and Kehn-Hall, K. (2013). Nuclear import and export inhibitors alter capsid protein distribution in mammalian cells and reduce Venezuelan Equine Encephalitis Virus replication. Antiviral Res. 100, 662–672.10.1016/j.antiviral.2013.10.004Search in Google Scholar
Noske, A., Weichert, W., Niesporek, S., Roske, A., Buckendahl, A.C., Koch, I., Sehouli, J., Dietel, M., and Denkert, C. (2008). Expression of the nuclear export protein chromosomal region maintenance/exportin 1/Xpo1 is a prognostic factor in human ovarian cancer. Cancer 112, 1733–1743.10.1002/cncr.23354Search in Google Scholar
Ossareh-Nazari, B., Gwizdek, C., and Dargemont, C. (2001). Protein export from the nucleus. Traffic 2, 684–689.10.1034/j.1600-0854.2001.21002.xSearch in Google Scholar
Panchal, M., Rawat, K., Kumar, G., Kibria, K.M., Singh, S., Kalamuddin, M., Mohmmed, A., Malhotra, P., and Tuteja, R. (2014). Plasmodium falciparum signal recognition particle components and anti-parasitic effect of ivermectin in blocking nucleo-cytoplasmic shuttling of SRP. Cell Death. Dis. 5, e994.10.1038/cddis.2013.521Search in Google Scholar
Pientka, F.K., Hu, J., Schindler, S.G., Brix, B., Thiel, A., Joehren, O., Fandrey, J., Berchner-Pfannschmidt, U., and Depping, R. (2012). Oxygen sensing by prolyl-4-hydroxylase PHD2 within the nuclear compartment and the influence of compartimentalisation on HIF-1 signalling. J. Cell Sci. 125, 5168–5176.10.1242/jcs.109041Search in Google Scholar
Ranganathan, P., Yu, X., Na, C., Santhanam, R., Shacham, S., Kauffman, M., Walker, A., Klisovic, R., Blum, W., Caligiuri, M., et al. (2012). Preclinical activity of a novel CRM1 inhibitor in acute myeloid leukemia. Blood 120, 1765–1773.10.1182/blood-2012-04-423160Search in Google Scholar
Ratcliffe, P.J. (2013). Oxygen sensing and hypoxia signalling pathways in animals: the implications of physiology for cancer. J. Physiol. 591, 2027–2042.10.1113/jphysiol.2013.251470Search in Google Scholar
Semenza, G.L. (2013). HIF-1 mediates metabolic responses to intratumoral hypoxia and oncogenic mutations. J. Clin. Invest. 123, 3664–3671.10.1172/JCI67230Search in Google Scholar
Semenza, G.L., Agani, F., Feldser, D., Iyer, N., Kotch, L., Laughner, E., and Yu, A. (2000). Hypoxia, HIF-1, and the pathophysiology of common human diseases. Adv. Exp. Med. Biol. 475, 123–130.10.1007/0-306-46825-5_12Search in Google Scholar
Steinhoff, A., Pientka, F.K., Mockel, S., Kettelhake, A., Hartmann, E., Kohler, M., and Depping, R. (2009). Cellular oxygen sensing: importins and exportins are mediators of intracellular localisation of prolyl-4-hydroxylases PHD1 and PHD2. Biochem. Biophys. Res. Commun. 387, 705–711.10.1016/j.bbrc.2009.07.090Search in Google Scholar
Stiehl, D.P., Jelkmann, W., Wenger, R.H., and Hellwig-Burgel, T. (2002). Normoxic induction of the hypoxia-inducible factor 1α by insulin and interleukin-1β involves the phosphatidylinositol 3-kinase pathway. FEBS Lett. 512, 157–162.10.1016/S0014-5793(02)02247-0Search in Google Scholar
Stommel, J.M., Marchenko, N.D., Jimenez, G.S., Moll, U.M., Hope, T.J., and Wahl, G.M. (1999). A leucine-rich nuclear export signal in the p53 tetramerization domain: regulation of subcellular localization and p53 activity by NES masking. EMBO J. 18, 1660–1672.10.1093/emboj/18.6.1660Search in Google Scholar
Tay, M.Y., Fraser, J.E., Chan, W.K., Moreland, N.J., Rathore, A.P., Wang, C., Vasudevan, S.G., and Jans, D.A. (2013). Nuclear localization of dengue virus (DENV) 1-4 non-structural protein 5; protection against all 4 DENV serotypes by the inhibitor Ivermectin. Antiviral Res. 99, 301–306.10.1016/j.antiviral.2013.06.002Search in Google Scholar
Vandromme, M., Gauthier-Rouviere, C., Lamb, N., and Fernandez, A. (1996). Regulation of transcription factor localization: fine-tuning of gene expression. Trends Biochem. Sci. 21, 59–64.10.1016/S0968-0004(96)80182-4Search in Google Scholar
Vaupel, P., Thews, O., and Hoeckel, M. (2001). Treatment resistance of solid tumors: role of hypoxia and anemia. Med. Oncol. 18, 243–259.10.1385/MO:18:4:243Search in Google Scholar
Wagstaff, K.M., Rawlinson, S.M., Hearps, A.C., and Jans, D.A. (2011). An AlphaScreen®-based assay for high-throughput screening for specific inhibitors of nuclear import. J. Biomol. Screen. 16, 192–200.10.1177/1087057110390360Search in Google Scholar
Wagstaff, K.M., Sivakumaran, H., Heaton, S.M., Harrich, D., and Jans, D.A. (2012). Ivermectin is a specific inhibitor of importin α/β-mediated nuclear import able to inhibit replication of HIV-1 and dengue virus. Biochem. J. 443, 851–856.10.1042/BJ20120150Search in Google Scholar
Wang, G.L., Jiang, B.H., Rue, E.A., and Semenza, G.L. (1995). Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc. Natl. Acad. Sci. USA 92, 5510–5514.10.1073/pnas.92.12.5510Search in Google Scholar
Yoneda-Kato, N. and Kato, J.Y. (2008). Shuttling imbalance of MLF1 results in p53 instability and increases susceptibility to oncogenic transformation. Mol. Cell Biol. 28, 422–434.10.1128/MCB.02335-06Search in Google Scholar
Supplemental Material
The online version of this article (DOI: 10.1515/hsz-2015-0171) offers supplementary material, available to authorized users.
©2015 by De Gruyter