studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 621A
bated cleaved caspase-3 enzyme activity and overexpression
of pJNK and RIP3 was characteristic of liver explants from
PBC patients compared with healthy controls, accompanied
by elevated protein levels of TNF and IL-6. Casp8 Δhepa after
BDL displayed decreased number and size of necrotic foci
compared with Casp8 f/f mice. Significantly decreased serum
alanine (ALT) and aspartate (AST) transaminases, TUNEL
staining, and cleaved Caspase-3 and cytochrome C protein
overexpression were found in Casp8 Δhepa mice 28 days after
BDL, along with diminished compensatory proliferation (Ki-67,
PCNA) and ductular reaction (CK-19). These results were translated
into a decreased inflammatory profile elicited by lower
IL-6, TNF-α protein levels and reduced infiltration of F4/80 + ,
Cd11b + and CD45 + populations. Overall, liver fibrogenesis
(Sirius red, Collagen IA1, ASMA) was significantly ameliorated
in Casp8 Δhepa . Mechanistically, decreased activation of
JNK, RIP1 and RIP3 correlated with the protection shown after
chronic BDL. Conclusions: CASP8 in hepatocytes is an essential
orchestrator of cholestatic liver injury and its specific modulation
might be an interesting pharmacologic target that can be
used in the clinic as treatment for obstructive liver disease.
Disclosures:
Christian Trautwein - Grant/Research Support: BMS, Novartis, BMS, Novartis;
Speaking and Teaching: Roche, BMS, Roche, BMS
The following authors have nothing to disclose: Francisco Javier Cubero, Jin
Peng, Maximilian Hatting, Gang Zhao, Miguel Eugenio Zoubek, Ricardo
Macías-Rodríguez, Astrid Ruiz-Margáin, Johanna Reissing, Henning W. Zimmermann,
Nikolaus Gassler, Tom Luedde, Christian Liedtke
829
NHERF-1 assembles macromolecular complexes in the
liver that are essential for the inflammatory response in
cholestasis
Man Li 1 , Albert Mennone 1 , Carol J. Soroka 1 , Lee R. Hagey 3 ,
Kathy M. Harry 1 , Edward J. Weinman 2 , James L. Boyer 1 ; 1 Internal
Medicine, Yale University School of Medicine, New Haven, CT;
2 University of Maryland School of Medicine, Baltimore, MD; 3 University
of California, San Diego, La Jolla, CA
The Na+/H+ exchanger regulatory factor 1 (NHERF-1/EBP50)
is a PDZ protein that has been shown to participate in signal
transduction by interacting with signaling molecules such as G
protein-coupled receptors as well as regulatory proteins such
as protein kinases. Our previous studies showed that deletion
of NHERF-1 in mice leads to reduced hepatic expression of
cytoskeletal ezrin-radixin-moesin (ERM) proteins and intercellular
adhesion molecule-1 (ICAM-1), a molecule that plays a key
role in neutrophil-mediated liver injury in mice after bile duct
ligation (BDL). NHERF-1-/- BDL mice have significantly lower
scores of hepatic necrosis, serum ALT, as well as reduced neutrophil
accumulation in the liver compared with the wild-type
(WT) BDL group. Aims: To further investigate the mechanisms
of protection of liver injury induced by BDL in NHERF-1-/- mice.
METHODS: Co-Immunoprecipitation was performed to identify
protein complexes that are formed with NHERF-1 in the liver
of sham and 7 day BDL WT mice. Immunoblotting was utilized
to compare protein expression in sham and BDL WT and
NHERF-1-/- mouse liver. Hepatic bile acids were analyzed
by mass spectrometry as well. RESULTS: NHERF-1 co-immunoprecipitated
with ICAM-1 in mouse liver, assembling ERM
proteins, ICAM-1 and F-actin into a macromolecule complex
that is increased in mouse liver and participates in neutrophil
infiltration after BDL. In the liver, NHERF-1 also interacts with
PKCζ, an atypical PKC that has been shown to phosphorylate
and activate NF-κB p65. Compared with the WT control,
NHERF-1-/- mice have reduced expression of hepatic PKCζ.
Expression of total NF-κB p65 as well as phosphorylated NF-κB
p65 was significantly reduced in the liver of NHERF-1-/- BDL
mice compared with WT BDL mice. While total bile acid concentrations
in the serum and liver of sham and BDL NHERF-1-
/- mice were not significantly different from the WT controls,
hepatic tetrahydroxylated bile acids and Cyp3a11 mRNA levels
were significantly higher in NHERF-1-/- BDL mice, indicating
a more profound adaptive response of the hepatocytes to
BDL in these mice. CONCLUSIONS: NHERF-1 participates in the
inflammatory and adaptive responses that is associated with
BDL induced liver injury. Deletion of NHERF-1 in mice leads
to disruption of the formation of ICAM-1-ERM-NHERF-1 and
PKCζ/NHERF-1 complexes, causing reduction of hepatic ERM
proteins, ICAM-1 and PKCζ, molecules that are essential for
the inflammatory response in cholestasis. Further study of the
role of NHERF-1 in the inflammatory response in cholestasis
and other forms of liver injury should lead to discovery of new
therapeutic targets in hepatic inflammatory diseases.
Disclosures:
James L. Boyer - Advisory Committees or Review Panels: Pfizer; Consulting:
abbvie
The following authors have nothing to disclose: Man Li, Albert Mennone, Carol J.
Soroka, Lee R. Hagey, Kathy M. Harry, Edward J. Weinman
830
Knockdown of α7-Nicotinic Receptor Inhibits Biliary
Proliferation and Hepatic Fibrosis during Extrahepatic
Cholestasis
Allyson Martinez 1 , April O’Brien 1 , Laurent Ehrlich 1 , David E. Dostal
1 , Shannon S. Glaser 1,2 ; 1 Department of Medicine, Texas A&M
Health Science Center and Central Texas Veterans Health Care
System, Temple, TX; 2 Scott & White Digestive Disease Research
Center, Temple, TX
In cholestatic liver diseases, cholangiocytes, through the products
of their cellular activation, are implicated as the key link
between bile duct injury and the subepithelial fibrosis that
characterizes chronic hepatobiliary injury. We have previously
demonstrated that activation of α7-nicotine receptor (nAChR)
with nicotine stimulates cholangiocyte proliferation that was
associated with increased profibrotic gene expression by cholangiocytes
and deposition of collagen in portal areas in normal
wild-type mice. We have also demonstrated that mechanical
stress, which occurs due to increased biliary pressure during
extrahepatic cholestasis, stimulates cholangiocyte proliferation.
The role of the α7-nAChR during extrahepatic cholestasis or
mechanical stress has not been thoroughly explored. Thus, the
AIM of our study was to determine the role of α7-nAChR in the
regulation of biliary proliferation and hepatic fibrosis during
extrahepatic cholestasis induced by bile duct ligation (BDL).
Methods: Studies were performed in normal and BDL (1 week)
wild-type (WT) and α7-nAChR knockout (KO) mice. Intrahepatic
bile duct mass (IBDM) and biliary proliferation was evaluated
by immunohistochemistry for CK-19 and PCNA in liver
sections and by immunoblots in isolated cholangiocytes. Liver
fibrosis was evaluated by Sirius red staining in liver sections
and by qPCR for the profibrotic markers [collagen 1 (COL1A1),
fibronectin (FN-1), and αSMA] in isolated cholangiocytes and
total liver samples. In vitro, mouse primary cholangiocytes were
plated on BioFlex culture plates and static equiaxial strain was
applied to the cells for 24 hrs in the presence and absence of
shRNA to knockdown α7-nAChR and the α7-nAChR antagonist
methyllycaconitine (1.4 nM) . Proliferation was evaluated by
immunoblots for PCNA and the expression of COL1A1, FN-1
and αSMA by qPCR. Results: In vivo, there was a significant
reduction in biliary proliferation (PCNA) and IBDM (CK-19)
in BDL α7-nAChR KO mice compared to BDL WT. In addi-