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Induction of cytokine production in cholesteatoma keratinocytes by extracellular high-mobility group box chromosomal protein 1 combined with DNA released by apoptotic cholesteatoma keratinocytes

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Abstract

High-mobility group box chromosomal protein 1 (HMGB-1), a nuclear DNA binding protein, was recently rediscovered as a new proinflammatory cytokine. The purpose of this study was to determine HMGB-1 expression in vivo and to identify the effect of extracellular HMGB-1 in inflammatory process associated with bone destruction in cholesteatoma. We investigated the expression and location of HMGB-1 in the cholesteatoma and healthy skin using an immunofluorescence assay. We also detected apoptosis and DNA fragments in the cholesteatoma by TUNEL staining. HMGB-1 concentration in apoptotic supernatants from UV light-treated cells, culture supernatants and its translocation in cholesteatoma keratinocytes stimulated by supernatants from UV light-treated cells were measured by immunoblot analysis and immunofluorescence assay. Cultures of human cholesteatoma keratinocytes were exposed to CpG-DNA, HMGB-1, or CpG-DNA complexed to HMGB-1 for 24 h. Cytokines in the culture supernatant were measured by ELISA. In addition, levels of proinflammatory cytokines released by cholesteatoma keratinocytes stimulated by supernatants from UV light-treated cells with or without anti-HMGB-1 antibodies and supernatants from UV light-treated cells with DNase 1 were measured by enzyme-linked immunosorbent assay. The expression of HMGB-1 in cholesteatoma increased and it translocated both to the cytoplasm and extracellular space. Furthermore, the HMGB-1 concentration in supernatants increased significantly after addition of supernatants from UV light-treated cells. TNF-α and IL-1β can be induced by purified HMGB-1 combined with CpG-DNA in the cholesteatoma keratinocytes. In addition, supernatants of apoptotic cells containing HMGB-1–DNA were effective in inducing TNF-α and IL-1β secretion. This study suggested that persistent expression of extracellular HMGB-1 and DNA fragments in cholesteatoma leads to TNF-α and IL-1β production, causing bone resorption and destruction. Thus, we have implicated that HMGB-1–DNA complexes might act as a key molecule involved in bone resorption associated with cholesteatoma.

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Acknowledgments

This work was supported by the Shanghai Shen-kang Medical Science Project SHDC12010119, Natural Science Foundation Project of CQ CSTC and Ministry Clinical Disciplines Project 2010-439.

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The authors declare that they have no competing interests.

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Authors and Affiliations

  1. Department of Otolaryngology, Eye & ENT Hospital, Fudan University, 83 Fenyang Road, Shanghai, China

    Zhangcai Chi, Zhengmin Wang, Qiong Liang, Yaying Zhu & Qiang du

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  1. Zhangcai Chi
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  2. Zhengmin Wang
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  3. Qiong Liang
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  4. Yaying Zhu
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  5. Qiang du
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Correspondence to Zhengmin Wang.

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Chi, Z., Wang, Z., Liang, Q. et al. Induction of cytokine production in cholesteatoma keratinocytes by extracellular high-mobility group box chromosomal protein 1 combined with DNA released by apoptotic cholesteatoma keratinocytes. Mol Cell Biochem 400, 189–200 (2015). https://doi.org/10.1007/s11010-014-2275-0

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  • DOI: https://doi.org/10.1007/s11010-014-2275-0

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