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Role of Ca/CaN/NFAT signaling in IL-4 expression by splenic lymphocytes exposed to phthalate (2-ethylhexyl) ester in spleen lymphocytes

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Abstract

The aims of present study were to investigate the effect of phthalate (2-ethylhexyl) ester (DEHP) and mono-(2-ethylhexyl) phthalate (MEHP) on Th1/Th2 balance signaling for interleukin 4 (IL-4) expression in splenic lymphocytes, and contribution of MEHP to any hypothesized changes in vitro. Primary splenic lymphocytes were exposed to DEHP/MEHP. ELISA and Western blotting were used to detect proteins. Confocal-microscopy was used to examine nuclear translocation. Nuclear factor of activated T cells (NFAT) DNA binding activity was examined by electrophoretic mobility-shift assay. DEHP significantly increased IL-4 and interferon gamma (IFN-γ) level, and reduced Th1/Th2 ratio (reflected by IFN-γ/IL-4) with 5 μg/L Concanavalin A (ConA) treatment. While MEHP reduced Th1/Th2 ratio (represented by IFN-γ/IL-6). IL-4 mRNA was significantly increased by DEHP but not by MEHP after PMA and Ion treatment. DEHP significantly inhibited NFATp protein in cytosol and nucleus. DEHP augmented nuclear translocation of NFATc in transfected EL4 cells and NFAT DNA-binding activity. DEHP-mediated enhancement of calcium-dependent phosphatase calcineurin (CaN) protein, and NFAT and IL-4 expression were abrogated by calcium antagonist verapamil and CaN inhibitor tarcolimus. Ca2+/calmodulin antagonist chlorpromazine significantly suppressed IL-4 and CaN production with no NFAT mRNA change. Our study suggests that DEHP and MEHP impact Th1/Th2 balance by modulating different cytokines. DEHP-affected IL-4 expression through Ca/CaN/NFAT signaling pathway, but no effect was discovered for MEHP.

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Acknowledgments

This research was supported by the National Science Foundation of China (NSFC) (Grant no. 30700671), the Research Foundation of Shenyang Medical College (20091008) and the National Science Foundation of China (NSFC) (Grant no. 81373029). We thank Dr. X. M. Gao (Health Science Center, Peking University) for preparing EL4 cells for us.

Conflict of interest

The authors report no conflict of interest related to this study or the findings specified in this paper.

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Authors and Affiliations

  1. Department of Toxicology, School of Public Health, Shenyang Medical College, Shenyang, 110034, Liaoning, China

    Xiucong Pei, Zhiwen Duan, Mingyue Ma & Yuming Zhang

  2. Science Experimental Center, Shenyang Medical College, Shenyang, 110034, Liaoning, China

    Li Guo

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  1. Xiucong Pei
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  2. Zhiwen Duan
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Correspondence to Xiucong Pei.

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Pei, X., Duan, Z., Ma, M. et al. Role of Ca/CaN/NFAT signaling in IL-4 expression by splenic lymphocytes exposed to phthalate (2-ethylhexyl) ester in spleen lymphocytes. Mol Biol Rep 41, 2129–2142 (2014). https://doi.org/10.1007/s11033-014-3062-4

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