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

Troglitazone is a synthetic ligand of peroxisome proliferators activated receptor-gamma (PPAR gamma) 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-alpha (AMPK alpha) 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 AMPK alpha 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 PPAR gamma activity by the irreversible antagonist GW9662 or by overexpressing dominate-negative PPAR gamma did not affect LC3-II accumulation and AMPK phosphorylation. Taken together, we demonstrate that autophagy promoted via troglitazone is correlated with AMPK alpha activation and independent of PPAR gamma activation and EGFR transactivation.