studies

218A AASLD ABSTRACTS HEPATOLOGY, October, 2015
Inflammasomes are new molecular platforms that respond to
microbial products through the synthesis of pro-inflammatory
cytokines. The Nlrp3 inflammasome activation is known to
determine Il-1β and Il-18 synthesis and release, thereby modulating
cell biology and eliciting pro-inflammatory signals in
different cell types. We sought to verify whether the inflammasome
is activated in sclerosing cholangitis and which are its
effects in reactive cholangiocytes. Methods: Expression levels
of the NLRP3 inflammasome were tested in cholangiocytes of
normal and cholestatic livers. The effects of Nlrp3 activation
induced by incubation with LPS/ATP were studied in vitro in
normal and siRNA Nlrp3 knockdown cholangiocytes. In vivo,
wild type (WT) and Nlrp3 A350VneoR
(Nlrp3 -/- ) mice were fed
with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC, a model
of sclerosing cholangitis) for 4 weeks. Results: Expression of
Nlrp3 and its components is increased in in cholangiocytes
of mice subjected to DDC and in patients affected by PSC
compared to normal conditions. LPS/ATP-induced activation
of Nlrp3 in cholangiocytes stimulates the expression of Il-18
and Il-6, but not of Il-1β. Nlrp3 activation also significantly
decreased the expression of Zonulin-1 and E-cadherin but
had no effect on cholangiocyte proliferation. The effects of
LPS/ATP-induced activation of Nlrp3 in cholangiocytes were
neutralized by knockdown of Nlrp3 by siRNA. In vivo, the
increases in the liver CK-19-positive parenchyma induced by 4
week DDC in WT animals were reduced in Nlrp3 -/- mice and
expression of Zonulin-1 tended to be re-established. Conclusions:
Nlrp3 is expressed in reactive cholangiocytes, both in
murine models and in PSC. The activation of Nlrp3 leads to
the synthesis of pro-inflammatory cytokines and influences the
epithelial integrity of cholangiocytes. These findings suggest
that microbial products may participate to the development of
certain cholangiopathies by activating the inflammasome in
cholangiocytes and altering the barrier function of the biliary
epithelium.
Disclosures:
The following authors have nothing to disclose: Luca Maroni, Laura Agostinelli,
Stefania Saccomanno, Eleonora Mingarelli, Chiara Rychlicki, Samuele De Minicis,
Jesus M. Banales, Antonio Benedetti, Gianluca Svegliati Baroni, Marco
Marzioni
22
Taurocholate Activates YAP via Sphingosine-1 Phosphate
Receptor 2 in Cholangiocytes
Kesong Peng 1 , Yunzhou Li 2 , Runping Liu 1,2 , Jing Yang 1 , Yongqing
Wang 3,1 , Guang Liang 4 , Phillip B. Hylemon 1,2 , Huiping Zhou 1,2 ;
1 Microbiology and Immunology, Virginia Commonwealth University,
Richmond, VA; 2 McGuire Veterans Affairs Medical Center,
Richmond, VA; 3 Nanjing Medical University, Nanjing, China;
4 School of Pharmacy, Wenzhou Medical University, Wenzhou,
China
Introduction: The transcription co-activators YAP/TAZ are the
core components of the Hippo pathway. Recent studies demonstrated
that activation of several G protein coupled receptors
(GPCRs), including sphingosine-1-phosphate receptor 2
(S1PR2), causes dephosphorylation and nuclear translocation
of YAP. Cholangiocytes are continuously exposed to high concentrations
of conjugated bile acids. Obstruction of bile flow
is a potent stimulus for cholangiocyte proliferation. We have
recently reported that taurocholate (TCA) promotes cholangiocyte
proliferation via activation of S1PR2, which is the predominant
S1P receptor in cholangiocytes. Recently, it was reported
that bile acids promote liver carcinogenesis through activation
of YAP. However, whether activation of S1PR2 by conjugated
bile acids induces YAP activation in cholangiocytes has not
been examined and is the focus of this study Methods: A mouse
cholangiocyte cell line was used for in vitro studies. Bile duct
ligation (BDL) mouse models were used to examine the role
of S1PR2 in YAP activation in vivo. A chemical antagonist of
S1PR2, JTE-013, was used to inhibit S1PR2 activation. S1P
was used as positive control. YAP activation was determined
by Western blot analysis. The mRNA levels of the YAP target
genes were determined by real-time RT-PCR. Results: Both
TCA- and S1P-induced cell migration and proliferation were
inhibited by JTE-013 in cholangiocytes. TCA- and S1P-induced
activation of YAP was indicated by decreasing of phosphorylated
YAP in cytosol and increasing of nuclear translocation of
YAP, which was markedly inhibited by JTE-013. In addition,
TCA and S1P also significantly up-regulated the expression
of the down-stream target genes of YAP including: IQGAP1,
cyclin D1, p21 and CTGF (connective tissue growth factor),
in cholangiocytes. YAP activation was significantly reduced in
BDL S1PR2 -/- mice as compared to wild-type mice. Discussion/
Conclusion: Elevated conjugated bile acid levels are correlated
with cholangiocyte proliferation and cholangiocarcinoma by
unknown mechanism(s). YAP-activation has been linked to
tumour growth. This study identified TCA/S1PR2/YAP/TAZ as
a cell signalling pathway involved in TCA-mediated cholangiocyte
proliferation and possibly transformation.
Disclosures:
The following authors have nothing to disclose: Kesong Peng, Yunzhou Li, Runping
Liu, Jing Yang, Yongqing Wang, Guang Liang, Phillip B. Hylemon, Huiping
Zhou
23
SRT1720 protects against cholestatic liver injury in mice
by altering hepatic bile acid composition and enhancing
urinary excretion of bile acids
Supriya Kulkarni 1 , Carol J. Soroka 1 , Lee R. Hagey 2 , James L.
Boyer 1 ; 1 Liver Center, Internal Medicine, Digestive Diseases, Yale
University School of medicine, New Haven, CT; 2 Division of Gastroenterology,
Department of Medicine, University of California at
San Diego, La Jolla, CA
BACKGROUND: Sirtuin1 (SirT1, mammalian homolog of S. Cerevisiae
enzyme Sir2) is a critical transcriptional and transactivational
regulator of murine Fxr. Liver specific knockout of Sirt1
deregulates Fxr activity. Our previous studies demonstrated that
bile duct ligation (BDL), or cholic acid (CA) feeding decrease
hepatic Sirt1 expression and that a Sirt1 activator SRT1720
improves cholestatic liver injury. AIM: To determine the mechanisms
by which Sirt1 activation may alleviate cholestatic liver
injury. METHODS: C57Bl/6 mice (n=11-13, male, 8-9 weeks
old) were fed standard rodent chow or chow supplemented
with 1% CA for 5 days. Both groups (n=5-6) were administered
either vehicle or Sirt1 activator, SRT1720 (50mg/kg/
day) for duration of the diet. Liver injury (ALT, ALP), BA levels in
plasma, liver, ileum, urine and the bile acid pool size as well
as gene and protein expression of BA transporter and synthesis
genes were determined. Immunoprecipitation assays were
used to determine Sirt1 acetylation and Fxr activity. RESULTS:
Sirt1 mRNA, protein and acetylation activity were significantly
reduced (75%) in CA fed mouse livers. SRT1720 administration
in CA fed mice reduced plasma ALT (40%) and plasma
BA (50%) levels while hepatic and total BA pool remained
unchanged. SRT1720 significantly increased tetra- hydroxylated
BA by 1.96x and decreased di-hydroxylated BA fraction
to 21% over CA fed mice and increased Cy2b10 (1.75x)
expression. SRT1720 increased ileal Fgf15 (30x) and hepatic
Fgfr4 (2X), decreased hepatic Cyp7a1 (99%), 27a1 (75%)
expression. SRT1720 also increased renal Mrp2 and Mrp4
expression (2-4.5x) along with increased urinary BA concen-