Oxidative stress induces the endoplasmic reticulum stress and facilitates inclusion formation in cultured cells.
OBJECTIVE
The precise mechanism of formation and significance of Mallory bodies (MBs) are poorly understood. The endoplasmic reticulum (ER) is the organelle responsible for proper folding and elimination of unfolded proteins. Therefore, failure of this function increases defective proteins in the cell.


METHODS
We examined the effects of oxidative stress on induction of ER stress and keratin 8 and 18 (K8/18)-containing inclusion formation in cultured human hepatoma cells and hepatocytes by immunofluorescence and immunoblot analyses.


RESULTS
Generation of H(2)O(2) was detected in glucose oxidase (GO)-treated cells by 2',7'-dichlorodihydrofluorescein diacetate and co-treatment with GO and acetyl-leucyl-leucyl-norleucinal (ALLN), a proteasome inhibitor, induced formation of extensive keratin inclusions that were inhibited by pre-treatment with N-acetyl-cysteine. These inclusions shared similar features with MBs by immunofluorescence analysis. Electron microscopy showed that these structures appeared near the nuclei, surrounded by filamentous structures. GO and ALLN upregulated the expression of ER stress markers, however, 4-phenylbutyrate, a chemical chaperone, reduced formation of inclusions and expression of the ER stress markers.


CONCLUSIONS
The oxidative stress coupled with limited inhibition of the proteasome induces dysfunction of the ER and results in inclusion formation in cultured cells. This suggests that ER stress plays a role in MB formation in liver disease.
Hanada
Shinichiro
S
Department of Medicine, Kurume University School of Medicine, and Research Center for Innovative Cancer Therapy, and Center of the 21st Century COE Program for Medical Science, Kurume University, Kurume, Japan. hanadas@med.kurume-u.ac.jp
Harada
Masaru
M
Kumemura
Hiroto
H
Bishr Omary
M
M
Koga
Hironori
H
Kawaguchi
Takumi
T
Taniguchi
Eitaro
E
Yoshida
Takafumi
T
Hisamoto
Takao
T
Yanagimoto
Chikatoshi
C
Maeyama
Michiko
M
Ueno
Takato
T
Sata
Michio
M
D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D005452:Fluoresceins / Q000494:pharmacology; D005949:Glucose Oxidase / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D053538:Keratin-18 / Q000032:analysis; D053533:Keratin-8 / Q000032:analysis; D007976:Leupeptins / Q000494:pharmacology; D008107:Liver Diseases / Q000378:metabolism* / Q000473:pathology; D018384:Oxidative Stress; D010654:Phenylbutyrates / Q000378:metabolism; D061988:Proteasome Inhibitors; D014407:Tumor Cells, Cultured
C110400:2',7'-dichlorodihydrofluorescein diacetate; D015853:Cysteine Proteinase Inhibitors; D005452:Fluoresceins; D053538:Keratin-18; D053533:Keratin-8; D007976:Leupeptins; D010654:Phenylbutyrates; D061988:Proteasome Inhibitors; C063261:acetylleucyl-leucyl-norleucinal; C075773:4-phenylbutyric acid; D006861:Hydrogen Peroxide; D005949:Glucose Oxidase