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Biochanin A impedes STAT3 activation by upregulating p38δ MAPK phosphorylation in IL-6-stimulated macrophages

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Abstract

Objective

IL-6-induced STAT3 activation is associated with various chronic inflammatory diseases. In this study, we investigated the anti-STAT3 mechanism of the dietary polyphenol, biochanin A (BCA), in IL-6-treated macrophages.

Methods

The effect of BCA on STAT3 and p38 MAPK was analyzed by immunoblot. The localization of both these transcription factors was determined by immunofluorescence and fractionation studies. The impact on DNA-binding activity of STAT3 was studied by luciferase assay. To understand which of the isoforms of p38 MAPK was responsible for BCA-mediated regulation of STAT3, overexpression of the proteins, site-directed mutagenesis, pull-down assays and computational analysis were performed. Finally, adhesion-migration assays and semi-quantitative PCR were employed to understand the biological effects of BCA-mediated regulation of STAT3.

Results

BCA prevented STAT3 phosphorylation (Tyr705) and increased p38 MAPK phosphorylation (Thr180/Tyr182) in IL-6-stimulated differentiated macrophages. This opposing modulatory effect of BCA was not observed in cells treated with other stress-inducing stimuli that activate p38 MAPK. BCA abrogated IL-6-induced nuclear translocation of phospho-STAT3 and its transcriptional activity, while increasing the cellular abundance of phospho-p38 MAPK. BCA-induced phosphorylation of p38δ, but not α, β, or γ was responsible for impeding IL-6-induced STAT3 phosphorylation. Interestingly, interaction with phospho-p38δ masked the Tyr705 residue of STAT3, preventing its phosphorylation. BCA significantly reduced STAT3-dependent expression of icam-1 and mcp-1 diminishing IL-6-mediated monocyte adhesion and migration.

Conclusion

This differential regulation of STAT3 and p38 MAPK in macrophages establishes a novel anti-inflammatory mechanism of BCA which could be important for the prevention of IL-6-associated chronic inflammatory diseases.

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Acknowledgements

This study was supported by the Department of Biotechnology, Govt. of India (Twining project: BT/469/NE/TBP/2013 & U-Excel project: BT/410/NE/U-Excel/2013). We also acknowledge the High-Performance Computing Centre, Tezpur University (India), for use of the PARAM Shavak. We thank Dr. Sougata Saha and Dr. Suman Dasgupta, Department of MBBT, Tezpur University, for their technical help.

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Authors and Affiliations

  1. Department of Molecular Biology and Biotechnology, Tezpur University, Napaam, Assam, 784028, India

    Anandita Basu, Anindhya Sundar Das & Rupak Mukhopadhyay

  2. Department of Food Engineering and Technology, Tezpur University, Tezpur, Assam, 784028, India

    Pallab Kumar Borah & Raj Kumar Duary

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  1. Anandita Basu
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  2. Anindhya Sundar Das
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Contributions

The experimental studies, data analyses, and manuscript preparation were performed by Anandita Basu and Anindhya Sundar Das. The computational study and data analysis were performed by Pallab Kumar Borah and Raj Kumar Duary. Experiment designing, data analyses, manuscript preparation, and fund acquisition were done by Rupak Mukhopadhyay. The final version of the manuscript was approved by all the authors.

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Correspondence to Rupak Mukhopadhyay.

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Studies on human peripheral blood mononuclear cells (PBMCs) were performed with due approval from the Tezpur University Ethics Committee (IEC No: 01/2014, dated 30/4/2014) with informed consent from healthy volunteers.

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Basu, A., Das, A.S., Borah, P.K. et al. Biochanin A impedes STAT3 activation by upregulating p38δ MAPK phosphorylation in IL-6-stimulated macrophages. Inflamm. Res. 69, 1143–1156 (2020). https://doi.org/10.1007/s00011-020-01387-1

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