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Dynamic localization and the associated translocation mechanism of HMGBs in response to GCRV challenge in CIK cells

Cellular & Molecular Immunology volume 12, pages 342–353 (2015)Cite this article

Abstract

High-mobility group box (HMGB) proteins, a family of chromatin-associated nuclear proteins, play amazingly multifaceted roles in the immune system of mammals. Thus far, little is known about the nucleocytoplasmic distribution of HMGBs in teleosts. The present study systematically investigated the dynamic localization of all six HMGB proteins in Ctenopharyngodon idella kidney (CIK) cells. Under basal conditions, all HMGBs exclusively localized to the nucleus. Grass carp reovirus (GCRV), polyinosinic–polycytidylic (poly(I∶C)) potassium salt and lipopolysaccharide (LPS) challenge evoked the nuclear export of HMGBs to various degrees: GCRV challenge induced the highest nuclear export of CiHMGB2b, and poly(I∶C) and LPS evoked the highest nucleocytoplasmic shuttling of CiHMGB1b. Overall, the nucleocytoplasmic shuttling of CiHMGB2a and CiHMGB3b was rarely induced by these challenges. Dynamic imaging uncovered that the nucleocytoplasmic GCRV-induced relocation of CiHMGB2b occurred in cells undergoing karyotheca rupture, apoptosis or proliferation. Western blot analyses were used to examine HMGB-EGFP fusion proteins in whole cell lysates, cytosol, nuclear fractions and culture medium. Further investigation demonstrated the nuclear retention of N-terminal HMG-boxes and the nucleocytoplasmic distribution of the C-terminal acidic tails. Comparative analyses of the dynamic relocation of full-length, truncated or chimeric HMGBs confirmed that the intramolecular interaction between HMG-boxes and C-tail domains mediated the nucleocytoplasmic translocation of HMGBs. These results not only provide an overall understanding of the subcellular localization of HMGBs, but also reveal the induction mechanism of the nucleocytoplasmic translocation of HMGBs by GCRV challenge, which lays a foundation for further studies on the interactions among pathogens, HMGBs and pattern recognition receptors in the innate immunity of teleosts.

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Acknowledgements

The authors thank Dr Chong Zhang for live cell imaging assistance and Professor Shantin Zhao and Dr Jiutao Wang for confocal microscopy assistance. This study was supported by the Program for New Century Excellent Talents in University (NCET-08-0466) and the Chinese Universities Scientific Fund (QN2009022).

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  1. College of Animal Science and Technology, Northwest A&F University, Yangling, China

    Youliang Rao, Jianguo Su, Chunrong Yang, Nana Yan, Xiaohui Chen & Xiaoli Feng

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Correspondence to Jianguo Su.

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Supplementary Information accompanies the paper on Cellular & Molecular Immunology's website (http://www.nature.com/cmi).

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Rao, Y., Su, J., Yang, C. et al. Dynamic localization and the associated translocation mechanism of HMGBs in response to GCRV challenge in CIK cells. Cell Mol Immunol 12, 342–353 (2015). https://doi.org/10.1038/cmi.2014.55

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