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Basic Research Paper

Autophagy augmented by troglitazone is independent of EGFR transactivation and correlated with AMP-activated protein kinase signaling

Jinguo Yan, Huaiyi Yang, Gang Wang, Lei Sun, Yuguang Zhou, Yinglu Guo, Zhijun Xi and Xuejun Jiang

Troglitazone is a synthetic ligand of peroxisome proliferators activated receptor-γ (PPARγ) and induces apoptosis in a variety of malignant cells. However, the underlying mechanism of its regulatory role in macroautophagy (hereafter autophagy) remains largely unknown. Using fluorescence and electron microscopy, we observed that autophagosomes could be induced and identified upon troglitazone challenge in both primary and epidermal growth factor receptor (EGFR)-expressed porcine aortic endothelial (PAE) cells. We report here that troglitazone augments AMP-activated protein kinase-α (AMPKα) phosphorylation, reduces p70S6 kinase phosphorylation and stimulates autophagy that is independent of EGFR expression and transactivation. Troglitazone stimulus reduced neither lysosomal staining nor GFP-LC3 dots of HeLa cells, when the cells pretreated with AG1478, a specific EGFR kinase inhibitor. Furthermore, AG1478 additively enhanced the troglitazone-induced degradation of sequestosome 1 (SQSTM1/p62), which is a selective substrate of autophagy. Inhibition of AMPKα activity either by compound C or by RNA interference markedly reduced the accumulation of microtubule-associated protein 1 light chain 3-II (LC3-II), a good indicator of autophagy; whereas blockage of PPARγ activity by the irreversible antagonist GW9662 or by over-expressing dominate-negative PPARγ did not affect LC3-II accumulation and AMPK phosphorylation. Taken together, we demonstrate that autophagy promoted via troglitazone is correlated with AMPKα activation and independent of PPARγ activation and EGFR transactivation.

Authors

Jinguo Yan

Biological Resource Center; The Institute of Microbiology; Graduate Schools; Chinese Academy of Sciences; Beijing, China

Huaiyi Yang

National Key Laboratory of Biochemical Engineering; Institute of Process Engineering; Chinese Academy of Sciences; Beijing, China

Gang Wang

The Institute of Urology; Peking University First Hospital; Beijing, China

Lei Sun

National Laboratory of Biomacromolecules; Center for Biological Electron Microscopy; Institute of Biophysics; Chinese Academy of Sciences; Beijing, China

Yuguang Zhou

Biological Resource Center; The Institute of Microbiology; Chinese Academy of Sciences; Beijing, China

Yinglu Guo

The Institute of Urology; Peking University First Hospital; Beijing, China

Zhijun Xi Corresponding author: xizhijun@hsc.pku.edu.cn

The Institute of Urology; Peking University First Hospital; Beijing, China

Xuejun Jiang

Biological Resource Center; The Institute of Microbiology; Chinese Academy of Sciences; Beijing, China