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Phosphorylated Heat Shock Protein 27 Inhibits Lipopolysaccharide-Induced Inflammation in Thp1 Cells by Promoting TLR4 Endocytosis, Ubiquitination, and Degradation

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Abstract

The aims of this study were to investigate the effect of Hsp27 on LPS-induced inflammation and identify the precise mechanisms about how Hsp27 regulates LPS-induced TLR4 signaling in Thp1 cells. Thp1 cells were transfected with Flag-Hsp27 or pcDNA3.1, and then treated with LPS for indicated time. TNF-α, IL-1β, and IL-6 were determined by ELISA. The protein levels of Hsp27, p-Hsp27 (Ser15, Ser78, and Ser82), and TLR4 were measured by Western blotting. In vitro study showed that over-expression of Hsp27 downregulated the release of TNF-α, IL-1β, and IL-6 and suppressed the activation of TLR4 signals after stimulated by LPS. The location of TLR4 and RAB5 was detected by confocal microscopy. Immunoprecipitation was used to determine the ubiquitination and degradation of TLR4 and interaction between Hsp27 and TLR4. Results showed that Hsp27 could promote TLR4 endocytosis and ubiquitination and degradation. Further research revealed that Hsp27 was phosphorylated after LPS, only phosphorylated Hsp27 can interact with TLR4 and inhibit the activation of TLR4 signaling, which was demonstrated by inhibition of Hsp27 phosphorylation with inhibitors or transfection of Hsp27 mutants into Thp1 cells. Phosphorylated Hsp27 reduced the release of TNF-α, IL-1β, and IL-6, and suppressed the activation of TLR4 signaling by promoting TLR4 endocytosis, ubiquitination, and degradation.

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Acknowledgments

This work was financially supported by grants from the Natural Science Foundation of China (Nos. 81671565, 81771703, and 81471557) and the Priority Academic Program Development of Jiangsu Higher Education Institution (PADD).

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  1. Jinfei Li and Xiaowen Qi contributed equally to this work.

Authors and Affiliations

  1. Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, College of Life Science, Nanjing Normal University, Nanjing, 210046, Jiangsu, People’s Republic of China

    Jinfei Li, Xiaowen Qi, Baolin Jiang, Ting Huang & Zhimin Yin

  2. State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, 210093, Jiangsu, People’s Republic of China

    Lan Luo

  3. Jurong Pepople’s Hospital, 60 West Street, Huayang Town, Jurong City, Zhenjiang, Jiangsu, 212400, People’s Republic of China

    Shixiang Liu

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Li, J., Qi, X., Jiang, B. et al. Phosphorylated Heat Shock Protein 27 Inhibits Lipopolysaccharide-Induced Inflammation in Thp1 Cells by Promoting TLR4 Endocytosis, Ubiquitination, and Degradation. Inflammation 42, 1788–1799 (2019). https://doi.org/10.1007/s10753-019-01041-x

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