Cholesterol depletion induces autophagy

https://doi.org/10.1016/j.bbrc.2006.10.042Get rights and content

Abstract

Autophagy is a mechanism to digest cells’ own components, and its importance in many physiological and pathological processes is being recognized. But the molecular mechanism that regulates autophagy is not understood in detail. In the present study, we found that cholesterol depletion induces macroautophagy. The cellular cholesterol in human fibroblasts was depleted either acutely using 5 mM methyl-β-cyclodextrin or 10–20 μg/ml nystatin for 1 h, or metabolically by 20 μM mevastatin and 200 μM mevalonolactone along with 10% lipoprotein-deficient serum for 2–3 days. By any of these protocols, marked increase of LC3-II was detected by immunoblotting and by immunofluorescence microscopy, and the increase was more extensive than that caused by amino acid starvation, i.e., incubation in Hanks’ solution for several hours. The induction of autophagic vacuoles by cholesterol depletion was also observed in other cell types, and the LC3-positive membranes were often seen as long tubules, >50 μm in length. The increase of LC3-II by methyl-β-cyclodextrin was suppressed by phosphatidylinositol 3-kinase inhibitors and was accompanied by dephosphorylation of mammalian target of rapamycin. By electron microscopy, autophagic vacuoles induced by cholesterol depletion were indistinguishable from those seen after amino acid starvation. These results demonstrate that a decrease in cholesterol activates autophagy by a phosphatidylinositol 3-kinase-dependent mechanism.

Section snippets

Materials and methods

Cells and antibodies. Human and mouse fibroblasts and Huh7 cells were cultured in Dulbecco’s modified Eagle’s medium (DME) supplemented with 10% fetal calf serum (FCS). Stable Huh7 cell lines expressing GFP-LC3 were generated by using a vector kindly provided by Dr. Tamotsu Yoshimori [13]. Rabbit anti-LC3 antibody was donated by Dr. Yasuo Uchiyama. Rat and mouse anti-LAMP1 antibodies (Developmental Studies Hybridoma Bank at the University of Iowa), rabbit anti-mTOR and rabbit anti-phospho-mTOR

Results and discussion

To induce autophagy by amino acid starvation, we cultured human fibroblasts in Hanks’ solution and monitored the relative ratio of LC3-II/LC-I by Western blot. LC3 is a mammalian homolog of Atg8 [15] and exists mostly as a soluble cytosolic protein, or LC3-I, at the normal culture condition. Upon induction of autophagy, LC3-I is modified with phosphatidylethanolamine, and incorporated into the autophagic membrane as LC3-II. By Western blotting, LC3-I and LC3-II are seen as bands at 18 kDa and 16 

Acknowledgments

We thank Drs. Tamotsu Yoshimori and Yasuo Uchiyama for providing precious reagents, Dr. Akitsugu Yamamoto for comments, and Mr. Tetsuo Okumura for technical assistance. This work was supported by Grants-in-Aid for Scientific Research and the 21st Century COE Program “Integrated Molecular Medicine for Neuronal and Neoplastic Disorders” of the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government.

References (26)

  • Y. Kondo et al.

    The role of autophagy in cancer development and response to therapy

    Nat. Rev. Cancer

    (2005)
  • S. Sarkar et al.

    Lithium induces autophagy by inhibiting inositol monophosphatase

    J. Cell Biol.

    (2005)
  • E.F. Blommaart et al.

    The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes

    Eur. J. Biochem.

    (1997)

    • Cited by (0)

    1

    These authors contributed equally to this work.

    View full text
    Copyright © 2006 Elsevier Inc. All rights reserved.