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

• Published in: Molecular and cellular biochemistry 2015-02, Vol.400 (1-2), p.189-200
• Main Authors: Chi, Zhangcai, Wang, Zhengmin, Liang, Qiong, Zhu, Yaying, du, Qiang
• Format: Article
• Language: English
• Subjects: Analysis; Apoptosis; Apoptosis - genetics; Biochemistry; Biomedical and Life Sciences; Bone Resorption - genetics; Bone Resorption - pathology; Cardiology; Cholesteatoma; Cholesteatoma - genetics; Cholesteatoma - pathology; Chromosomes; Cytokines; Deoxyribonucleic acid; DNA; DNA-Binding Proteins - genetics; Ear diseases; Enzyme-linked immunosorbent assay; Enzymes; Gene Expression Regulation; HMGB1 Protein - biosynthesis; HMGB1 Protein - genetics; HMGB1 Protein - metabolism; Humans; Interleukin-1beta - biosynthesis; Keratinocytes - metabolism; Keratinocytes - pathology; Life Sciences; Medical Biochemistry; Oncology; Protein binding; Proteins; Skin; Translocation; Tumor Necrosis Factor-alpha - biosynthesis; Ultraviolet radiation
• ISSN: 0300-8177; 1573-4919
• DOI: 10.1007/s11010-014-2275-0

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.