studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 615A
816
The bile-acid receptor TGR5 regulates biliary epithelium
permeability
José Ursic-Bedoya 1,2 , Hayat Simerabet 1,2 , Isabelle Doignon 1,2 ,
Noémie Péan 1,2 , Zahra Tanfin 1,2 , Christoph Ullmer 3 , Lydie Humbert
4,5 , Dominique Rainteau 4,5 , Doris Cassio 1,2 , Thierry Tordjmann
1,2 ; 1 U1174, INSERM, Orsay, France; 2 Université Paris sud,
Orsay, France; 3 Pharma Research and Early Development, Roche
Innovation Center Basel, Basel, Switzerland; 4 U1057, INSERM,
Paris, France; 5 UPMC, Paris, France
Background: TGR5, the bile acid (BA) G-protein-coupled receptor
protects the liver against BA overload during regeneration.
After partial hepatectomy, peribiliary hepatic necrosis occurs
in TGR5 KO but not in wild type (WT) mice, suggesting that
TGR5, highly expressed in biliary epithelial cells, regulates
biliary epithelium permeability. Methods: Trans-epithelial resistance
(TER) and FITC-dextran transfer were measured in the
NRC (Normal Rat Cholangiocyte) cell line cultured on transwell
inserts. To study biliary epithelial permeability in vivo in mice,
fluorescent dextran or a modified BA (Glycocholic acid) were
injected gallbladder (GB) lumen and traced (spectrofluorimetry
& Mass Spectrometry) in plasma and liver. Cells and mice
were stimulated with RO5527239, a TGR5 specific agonist,
and with taurolitocholic acid. TGR5-induced signaling pathways
were studied by western blot (WB), using selective inhibitors.
Tight junction (TJ) proteins expression was investigated by
qPCR, WB and immunofluorescence in NRC, in livers and GB
from WT and TGR5-KO mice. Results: In NRC, TGR5 agonists
significantly increased TER, reduced dextran passage, induced
ERK phosphorylation and EGFR transactivation. Inhibition of
cAMP production or MAPK pathways suppressed TGR5 agonists
effect on NRC permeability. In TGR5-KO as compared
with WT mice, although TJ proteins (ZO-1, occludin and JAM-
A) mRNA expression in GB was significantly reduced, only
JAM-A expression and localization at TJ were altered in bile
ducts and GB. TGR5 agonists induced JAM-A phosphorylation
and TJ localization in vitro in NRC, and in vivo after injection
in WT but not in TGR5-KO GB lumen. In vivo, BA and dextran
transepithelial transfer after GB injection was increased in
TGR5-KO as compared with WT mice (Fig.1). BA transporters
mRNA expression (ASBT, OSTb, MRP3) was similar in WT
and TGR5-KO GB, suggesting that TGR5 controls paracellular
rather than transcellular permeability. Conclusion: The BA
receptor TGR5 reinforces biliary epithelial sealing in vitro and
in vivo, likely through modulation of TJ protein expression and
phosphorylation, protecting liver parenchyma against bile leakage.
Disclosures:
Christoph Ullmer - Employment: F. Hoffmann-La Roche AG
The following authors have nothing to disclose: José Ursic-Bedoya, Hayat Simerabet,
Isabelle Doignon, Noémie Péan, Zahra Tanfin, Lydie Humbert, Dominique
Rainteau, Doris Cassio, Thierry Tordjmann
817
Knockout of the Secretin/Secretin Receptor Axis Delays
Regeneration of small and large cholangiocytes by
enhanced senescence following 70% partial hepatectomy
(PH)
Ying Wan 3 , Shannon S. Glaser 1 , Nan Wu 4 , Fanyin Meng 2 ,
Julie Venter 4 , Romina Mancinelli 5 , Heather L. Francis 2 , Antonio
Franchitto 5 , Paolo Onori 5 , Tina Kyritsi 4 , Shanika Avila 4 , Guido
Carpino 7 , Holly A. Standeford 6 , Gianfranco Alpini 2 , Eugenio
Gaudio 5 ; 1 Central Texas Veterans Health Care System and Texas
A&M HSC College of Medicine, Temple, TX; 2 Research, S&W
DDRC and Medicine, Veterans Health Care System and Texas
A&M HSC and BaylorScott&White, Temple, TX; 3 Operational
Funds, BaylorScott&White, Temple, TX; 4 Medicine, Texas A&M
University HSC, Temple, TX; 5 Department of Anatomical, Histological,
Forensic Medicine and Orthopedics Sciences, University La
Sapienza, Rome, Italy; 6 Research, Central Texas Veterans Health
Care System, Temple, TX; 7 Dept Health Sciences, University of
Rome “Foro Italico, Rome, Italy
Limited data exists regarding the neuroendocrine factors that
regulate the renewal of the biliary tree after PH. The secretin
(SCT)/secretin receptor (SR) axis is normally expressed only by
large cholangiocytes. However, during the damage of large
bile ducts small cholangiocytes (more resistant to injury) replenish
the large damaged ducts by de novo acquisition of large
biliary phenotypes. Cellular senescence is a state of irreversible
cell cycle arrest involved in biliary diseases including primary
sclerosing cholangitis and primary biliary cirrhosis. Large,
senescent cholangiocytes are the target cells in the cholestatic
models of bile duct ligation and MDR2 KO (PSC). Hypothesis:
in hepatectomized SCT/SR double knockout (DKO) mice there
is reduced regeneration of small and large bile ducts due to
enhanced senescence. Methods: The studies were performed
in normal wild type (WT) and SCT/SR DKO mice at 0, 3, 6
hours and 1, 3, 7, and 14 days after sham or 70% PH. Liver
injury was evaluated by: (i) H&E staining in liver sections; and
(ii) measurement of serum levels of transaminases. We evaluated
biliary proliferation by: (i) immunohistochemistry (IHC) for
PCNA and intrahepatic bile duct mass (IBDM) by CK-19 IHC in
liver sections; and (ii) qPCR for PCNA and CK-19 in total liver
samples and small and large cholangiocytes. Cellular senescence
was evaluated by SA-β-Gal staining in liver sections
and qPCR for P18, PAI-1 and CCl2 in total liver samples and
small and large cholangiocytes. Results: Small cholangiocytes
from PH WT mice displayed less senescent features compared
to large cholangiocytes. In PH WT mice, small less senescent
cholangiocytes regenerate as early as 1 hr and rebuild IBDM
within 1 day of PH, whereas large regenerate as early as 6 hr
and rebuild IBDM at a lower rate (after 3-7 days of PH). Large
cholangiocytes from DKO PH animals displayed increased
expression of senescent markers relative to PH WT mice. In
DKO PH mice, there was reduced regeneration time (by PCNA)
and regrowth (by CK-19) of small and large bile ducts concomitant
with enhanced expression of senescence markers (p18,
PAI-1 and CCL2) in large cholangiocytes as well as in small
cholangiocytes that display normally low levels of senescence.
In SCT/SR double KO mice, at day 14 of PH IBDM returned at
only 50% of the original biliary mass (0.238 ± 0.014, sham,
vs. 0.128 ± 0.007, SCT/SR KO, p