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Glucocorticoid Receptor β Acts as a Co-activator of T-Cell Factor 4 and Enhances Glioma Cell Proliferation

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Abstract

We previously reported that glucocorticoid receptor β (GRβ) regulates injury-mediated astrocyte activation and contributes to glioma pathogenesis via modulation of β-catenin/T-cell factor/lymphoid enhancer factor (TCF/LEF) transcriptional activity. The aim of this study was to characterize the mechanism behind cross-talk between GRβ and β-catenin/TCF in the progression of glioma. Here, we reported that GRβ knockdown reduced U118 and Shg44 glioma cell proliferation in vitro and in vivo. Mechanistically, we found that GRβ knockdown decreased TCF/LEF transcriptional activity without affecting β-catenin/TCF complex. Both GRα and GRβ directly interact with TCF-4, while only GRβ is required for sustaining TCF/LEF activity under hormone-free condition. GRβ bound to the N-terminus domain of TCF-4 its influence on Wnt signaling required both ligand- and DNA-binding domains (LBD and DBD, respectively). GRβ and TCF-4 interaction is enough to maintain the TCF/LEF activity at a high level in the absence of β-catenin stabilization. Taken together, these results suggest a novel cross-talk between GRβ and TCF-4 which regulates Wnt signaling and the proliferation in gliomas.

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Acknowledgments

This work was supported by the Natural Science Foundation of China (NFSC) grants (no. 81372710 and 81000527 to Jian Zou; no. 81101801 to Pei-Hua Lu; no. 81100547 to Jie Xiang); Natural Science Foundation of Jiangsu Province (NFSJS) grant (no. BK2010159 to Jian Zou). We thank Bert Vogelstein for having provided the pcDNA/Myc TCF4, pGL3-OT, and pGL3-OF plasmids, these plasmids were obtained through the Addgene plasmid depository. The authors thank Clarity Manuscript Consultants for their language editing.

Financial Support

This work was supported by Natural Science Foundation of China (NFSC) grants (no. 81372710 and 81000527 to Jian Zou; no. 81101801 to Peihua Lu; no. 81100547 to Jie Xiang); Natural Science Foundation of Jiangsu Province (NFSJS) grant (no. BK2010159 to Jian Zou).

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None.

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Authors and Affiliations

  1. Department of Clinical Laboratory Science, Wuxi People’s Hospital Affiliated to Nanjing Medical University, Wuxi, 214023, People’s Republic of China

    Qian Wang, Ping Xie, Ying Yin, Bin Zhang, Hui-Jun Mu, Wei-Zhen Qiao & Jian Zou

  2. Wuxi Institute of Translational Medicine, Wuxi, 214023, People’s Republic of China

    Qian Wang, Pei-Hua Lu, Jie Xiang, Ping Xie, Ying Yin, Bin Zhang, Hui-Jun Mu, Wei-Zhen Qiao & Jian Zou

  3. Jiangsu Key laboratory of Organ Transplantation, Wuxi, 214023, People’s Republic of China

    Qian Wang, Pei-Hua Lu, Jie Xiang, Ping Xie, Ying Yin, Bin Zhang, Hui-Jun Mu, Wei-Zhen Qiao & Jian Zou

  4. Department of Medical Oncology, Wuxi People’s Hospital Affiliated to Nanjing Medical University, Wuxi, 214023, People’s Republic of China

    Pei-Hua Lu

  5. Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai, 200040, People’s Republic of China

    Zhi-Feng Shi

  6. Molecular Biology & Protein Engineering, XBio, Inc., Shanghai, 201318, People’s Republic of China

    Yan-Juan Xu

  7. Training Center of Basic Medical Experiments, School of Medicine, Shanghai Jiaotong University, Shanghai, 200025, People’s Republic of China

    Yan-Xia Wang

  8. Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN, 46202, USA

    Ling-Xiao Deng

  9. Spinal Cord and Brain Injury Research Group, Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN, 46202, USA

    Ling-Xiao Deng

  10. Department of Neurosurgery, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200127, People’s Republic of China

    Hua Cui

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  1. Qian Wang
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Correspondence to Jian Zou.

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Qian Wang, Pei-Hua Lu, and Zhi-Feng Shi equally contributed to this work.

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Supplementary Fig. 1

GRβ knock-down enhanced GRα mediated trans-repression on TCF/LEF in U118 glioma cells. a A cell growth assay showed GRβ knock-down enhanced the growth inhibition of dexamethasone (Dex) in U118 cells. scGRβ-U118 and siGRβ-U118 cells were treated with vehicle or Dex (1 μM) every day and cultured for 3 days. The relative cell number of Dex treated scGRβ-U118 and siGRβ-U118 cells were compared. **p<0.01, n=6. b Luciferase reporter assays showed GRβ knock-down enhanced the inhibition of Dex on TCF/LEF transcriptional activity. scGRβ-U118 and siGRβ-U118 cells were transfected with an OT/OF-Luc Flash reporter and pRL-TK. After treatment with vehicle or Dex (1μM) for 3 days, luciferase activities were assayed. The normalized luciferase of Dex treated scGRβ-U118 and siGRβ-U118 cells were compared. **p<0.01, n=6. (GIF 12 kb)

High resolution image (TIFF 270 kb)

Supplementary Fig. 2

Cell growth assay showed that Mifepristone (RU486) inhibited cell growth of glioma cells. U118 (a) and Shg44 (b) glioma cells were treated with vehicle or RU486 (1μM) for different periods. *p<0.05, n=6. (GIF 8 kb)

High resolution image (TIFF 241 kb)

Supplementary Fig. 3

RU486 did not inhibit TCF/LEF activity in glioma cells. a U118 and Shg44 cells were transfected with an OT/OF-Luc Flash reporter and pRL-TK for 24 h. After treatment with vehicle or RU486 (1μM) for another 12 h, the luciferase activities were assayed. b IP results showed that RU486 had no effects on the interaction between TCF-4 and GRβ. Cells were treated with RU486 (1μM) or vehicle for 24 h. Cell proteins were immunoprecipitated with mouse anti-TCF-4 and analyzed by an IB with rabbit anti-GRβ and TCF-4. The IB of lysate showed that no change of GRβ expression occurred after the treatment with RU486. (GIF 11 kb)

High resolution image (TIFF 349 kb)

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Wang, Q., Lu, PH., Shi, ZF. et al. Glucocorticoid Receptor β Acts as a Co-activator of T-Cell Factor 4 and Enhances Glioma Cell Proliferation. Mol Neurobiol 52, 1106–1118 (2015). https://doi.org/10.1007/s12035-014-8900-9

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