A Study for the Autophagic Process in Human Umbilical Vein Endothelial Cells Stimulated with Bacteroides fragilis Enterotoxin

Abstract

ABSTRACT

A Study for the Autophgic Process in Human Umbilical Vein Endothelial Cells Stimulated with Bacteroides fragilis Enterotoxin

Su Hyuk Ko Dept. of Biomedical Science The graduate school Hanyang University

(Directed by Prof. Jung Mogg Kim)

Background Enterotoxigenic Bacteroides fragilis (ETBF) secretes a ~20-kDa metalloprotease toxin (BFT). BFT induces inflammatory signaling cascade in endothelial cells (ECs). Autophagy is a catabolic pathway that regulates homeostasis in various cells. Deregulation of autophagy can contribute to a broad spectrum of mammalian diseases. Although many studies have been carried out to elucidate signals related with autophagy, formation of autophagy in BFT-stimulated ECs is still unknown. This study was conducted to investigate the role of BFT as a regulator of autophagy in ECs.

Methods Human umbilical vein endothelial cells (HUVECs) were cultured in the presence or absence of BFT. Process of autophagy was assessed by western blot, immunofluorescence microscopy, and transmission electron microscopy. Signals of transcription factors were measured by electrophoretic mobility shift assays.

Results 1. Treatment of HUVECs with BFT increased formation of autophagosome and inhibited autophagosomal-lysosomal fusion. 2. BFT activated mTOR signaling, which is known as autophagy suppressor. 3. Activation of mTOR was not influence for BFT-induced inhibition of autophagosomal-lysosomal fusion. 4. NF-κB and AP-1 transcription factors were increased in BFT-stimulated HUVECs. 5. BFT led overexpression of CHOP in HUVECs. 6. BFT induces development of autolysosome when AP-1 and CHOP were inhibited by infection with lentivirus containing their dominant negative form or shRNA. 7. BFT-induced CHOP expression was decreased when AP-1 signal was blocked in HUVECs. 8. BFT evaluates cytosolic calcium levels in HUVECs. 9. Calcium channel opener suppresses BFT-induced arrest of autophagic process in HUVECs.

Conclusion These results suggest that BFT leads formation of autophagosome and inhibits autopagosomal-lysosomal fusion in BFT-exposed ECs. This process may be regulated by AP-1 and CHOP signaling and suppression of AP-1 and CHOP allows the fusion between autophagosome and lysosome.