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Molecular and Cellular Biochemistry

, Volume 420, Issue 1–2, pp 53–63 | Cite as

Silibinin negatively contributes to primary cilia length via autophagy regulated by histone deacetylase 6 in confluent mouse embryo fibroblast 3T3-L1 cells

  • Qian Xu
  • Wei Liu
  • Xiaoling Liu
  • Weiwei Liu
  • Hongju Wang
  • Guodong Yao
  • Linghe Zang
  • Toshihiko Hayashi
  • Shin-ichi Tashiro
  • Satoshi Onodera
  • Takashi IkejimaEmail author
Article

Abstract

Primary cilium is a cellular antenna, signalling as a sensory organelle. Numerous pathological manifestation is associated with change of its length. Although the interaction between autophagy and primary cilia has been suggested, the role of autophagy in primary cilia length is largely unknown. In this study the primary cilia were immunostained and observed by using confocal fluorescence microscopy, and we found that silibinin, a natural flavonoid, shortened the length of primary cilia, meanwhile it also induced autophagy in 3T3-L1 cells. This study was designed to investigate the significance of silibinin-induced autophagy in primary ciliary structure in confluent mouse embryo fibroblast 3T3-L1 cells. Either blocking the autophagic flux with pre-treatment with the autophagy inhibitor, 3-methyladenine (3-MA), or transfection of siRNA targeting LC3 inhibited the reduction of cilia length caused by silibinin exposure. Autophagy induced by silibinin decreased expressions of the cilia-associated proteins, such as IFT88, KIF3a and Ac-tubulin, while 3-MA restored it, indicating that autophagy induced by silibinin led to a reduction of primary cilia length. Histone deacetylase 6 (HDAC6), which was suggested as a mediator of autophagy, was up-regulated by silibinin in a time-dependent manner. In addition, 3T3-L1 cells treated with siRNA against HDAC6 had a reduced autophagic level and were protected from silibinin-induced cilia shortening. Taken together, we conclude that the HDAC6-mediated autophagy negatively regulates primary cilia length during silibinin treatment and has the potential to serve as a therapeutic target for primary cilia-associated ciliopathies. These findings thus provide new information about the potential link between autophagy and primary cilia.

Keywords

Autophagy Primary cilia Silibinin 3T3-L1 cells HDAC6 

Notes

Acknowledgments

This research was supported by National Natural Science Foundation of China (No. 81273517).

Supplementary material

11010_2016_2766_MOESM1_ESM.tif (28.3 mb)
Supplementary Figure 1 Silibinin influenced the induction of autophagy but not the lysosomal degradation process. (A) Effects of CQ on the expression of KIF3a, IFT88, Ac-tubulin, and the conversion from LC3 I to LC3 II were determined by western blot analysis. (B) Confluent 3T3-L1 cells were incubated with 50 μM silibinin in the presence or absence of CQ for 24 h and immunofluorescence staining of acetylated α-tubulin, LC3 and nuclei were stained with DAPI (blue). Scale bar, 10 μm. (TIFF 28934 kb)

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Qian Xu
    • 1
  • Wei Liu
    • 1
  • Xiaoling Liu
    • 1
  • Weiwei Liu
    • 1
  • Hongju Wang
    • 1
  • Guodong Yao
    • 1
  • Linghe Zang
    • 1
  • Toshihiko Hayashi
    • 1
  • Shin-ichi Tashiro
    • 2
  • Satoshi Onodera
    • 3
  • Takashi Ikejima
    • 1
    Email author
  1. 1.China–Japan Research Institute of Medical and Pharmaceutical SciencesPharmaceutical UniversityShenyangChina
  2. 2.Department of Medical Education and Primary CareKyoto Prefectural University of MedicineKyotoJapan
  3. 3.Department of Clinical and Pharmaceutical SciencesShowa Pharmaceutical UniversityTokyoJapan

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