studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 503A
tein levels were determined by quantitative PCR and western
blot analysis. Results: Six hours after 300 mg/kg of APAP
injection, serum ALT levels were 3831 and 7897 U/L and
relative hepatic necrosis area was 22.7 and 47.1% of view
field in wild-type and Chop-null mice, respectively. The Chopnull
mice showed a severer liver injury than the wild-type mice.
When phosphorylations of AKT, ERK, JNK, and p38 proteins
in the livers were investigated, APAP-enhanced JNK phosphorylation
was reduced in the Chop-null mice, compared to the
wild-type. In addition, MKK4 and JUN phosphorylations were
also reduced in Chop-null mice, suggesting that JNK signal
was attenuated in the Chop-null livers. Increased mRNA levels
of TIFA, an NF-kB activator, and enhanced phosphorylation of
FOS, an NF-kB-target protein, were observed in the livers of
the Chop-null mice, suggesting that NF-kB signal was accelerated
in the Chop-null livers. Hepatic ratio of LC3-II protein
to LC3-I protein, an indicator of autophagy, was increased in
the wild-type mice after the APAP injection (2.4 fold), but the
increase was diminished in the Chop-null mice. Furthermore
protein levels of hepatic p62 (also called SQSTM1), protein
decomposed under autophagy, were higher in the Chop-null
mice than that in the wild-type mice. There results strongly indicated
a view that autophagy signal was attenuated in the livers
of Chop-null mice Conclusions: In this study, molecular linkage
of CHOP-JNK-autophagy was identified as a protective factor
in APAP-induced liver injury. Although further study is required,
accelerated NF-kB signal in Chop-null livers may interfere with
the protective role of the JNK-autophagy.
Disclosures:
Norifumi Kawada - Grant/Research Support: BMS, Chugai, Kowa; Speaking
and Teaching: MSD, Janssen
The following authors have nothing to disclose: Tsutomu Matsubara, Yuga Teranishi,
Kazuki Nakatani, Kazuo Ikeda
593
The Effect of Treatment with Chemical Chaperones and
Zinc Acetate on Hepatocytes Treated with Excess Copper
Shinji Oe, Koichiro Miyagawa, Yuichi Honma, Masaru Harada;
School of Medicine, University of Occupational and Environmental
Health, Japan., Fukuoka, Japan
[Purpose] Copper is an essential trace element in human activity.
But excess copper gives harm to human activity. Wilson
disease is a genetic disorder characterized by excess copper
deposition in various organs. Excess copper-derived oxidants
contribute to progression of liver disease in Wilson disease.
Oxidative stress induces accumulation of abnormal proteins.
The endoplasmic reticulum (ER) is subcellular organelle which
plays the role of proper protein folding. Accumulation of misfolded
proteins disturbs ER homeostasis resulting in ER stress.
Zinc acetate has been commonly used to treat Wilson disease,
because it increases metallothionein production and inhibits
absorption of copper in the intestinal epithelia. The effects of
chemical chaperones and zinc acetate on copper-induced hepatotoxicity
have not been examined. In this study, we evaluated
the effects of chemical chaperone and zinc acetate on copper-induced
disruption of cell homeostasis. [Methods] We used
human hepatoma cell line (Huh7) and immortalized human
hepatocyte cell line (OUMS29). The following materials were
used: copper sulfate; acetyl-leucyl-leucyl-norleucinal and epoxomicin
as proteasome inhibitors (PIs); bathocuproine disulfonate
as copper chelator; n-acetyl-l-cysteine; zinc acetate; 4-phenylbutyrate
(PBA) and ursodeoxycholic acid (UDCA) as chemical
chaperones. We detected the reactive oxygen species using
2’,7’-dichlorodihydrofluorescein .
We analyzed copper-induced
ER stress by immunoblotting for ER stress markers, including
phospho α-subunit of eukaryotic initiation factor 2 and X-box
binding protein 1. Hepatocyte apoptosis treated with copper
with or without PI was determined by detection of cleaved poly
ADP-ribose polymerase and cleaved caspase 3 by immunoblotting
analysis and immunofluorescence staining, respectively.
Furthermore, we examined cell proliferation on excess copper
by immunofluorescence staining using Ki67. [Results] Excess
copper induced oxidative stress and ER stress. Chemical chaperones
and zinc acetate reduced copper-induced oxidative
stress and ER stress. Co-treatment of copper and PI exacerbated
copper-induced apoptosis. Although excess copper leaded to
reduction in cell proliferation, chemical chaperones rescued
the proliferation block in cells treated with copper. However
zinc acetate did not exert this effect. [Conclusions] Excess copper
induced hepatotoxicity. Chemical chaperones, PBA and
UDCA, and zinc acetate reduced hepatotoxicity. These results
suggest that chemical chaperones may have beneficial effects
for the treatment of Wilson disease. On the other hand, zinc
acetate used as the treatment of Wilson disease may inhibit
liver injury directly.
Disclosures:
The following authors have nothing to disclose: Shinji Oe, Koichiro Miyagawa,
Yuichi Honma, Masaru Harada
594
Sumoylation Regulates Lipopolysaccharide-induced
Macrophage Activation Modulating Ubiquiting conjugating
enzyme 9 Phosphorylation in Mouse Liver and
Kupffer Cells
Maria Lauda Tomasi 2,1 , Komal Ramani 2 , Minjung Ryoo 2 ; 1 Medicine,
University of Southern California, Los Angeles, CA; 2 Medicine,
Cedars-Sinai Medical Center, Los Angeles, CA
Propose of study: Activation of macrophages by lipopolysaccharide
(LPS), a conserved component of the Gram-negative
bacterium’s outer membrane, leads to production of a large
number of immunoregulatory molecules including tumor necrosis
factor α (TNF-α), interleukin 1β (IL-1β), interleukin 6 (IL-6),
arachidonic acid, as well as reactive oxygen species (ROS).
LPS level is increased in the blood of alcohol abusers and ethanol-fed
rodents. The small ubiquitin-related modifier (SUMO)
family of proteins is a more recently discovered posttranslational
modification. Cellular stress arising from ROS is a robust stimulus
for protein SUMOylation. SUMOylation is widely deemed
as a protective mechanism; loss of SUMO conjugation reduces
cell and organism viability. Ubiquitin conjugating enzyme 9
(UBC9) is the only SUMO E2 enzyme. We demonstrated that
UBC9 is phosphorylated by Cyclin-dependent kinase 1 (CDK1)
and is more stable when phosphorylated. Furthermore, we
recently found that LPS lowers UBC9 and SUMO expression
in RAW cells, nevertheless the mechanism is still unknown. The
aim of this study was to examine the role of Ubc9-mediated
sumoylation in LPS-activated macrophages and elucidate the
molecular mechanism(s). Methods: Studies were done using in
vivo LPS-treated mice, primary mouse Kupffer and RAW cells.
Real-time PCR and Western blotting measured mRNA and protein
levels, respectively. UBC9 phosphorylation was assessed
by phos-tagÔ SDS-PAGE. Results: LPS treatment increased ROS
production (H 2
O 2
) in RAW cells, while UBC9 knockdown alone
or co-treatment with LPS completely prevented LPS-induced
ROS production. UBC9 siRNA alone or with LPS co-treatment
induced apoptosis and increased IL-1β, iNOS, IL-6 and TNF-α
mRNA levels in RAW cells; however, when LPS and UBC9
siRNA were combined, they further raised these inflammatory
signals. We found that LPS treatment lowers UBC9 protein