studies

892A AASLD ABSTRACTS HEPATOLOGY, October, 2015
1393
Role of CREBH activation induced by HCV infection in
up-regulation of TGF-β2 expression and fibrogenesis
Takeshi Chida 1,2 , Masahiko Ito 1 , Kazuhito Kawata 2 , Yoshimasa
Kobayashi 2 , Tetsuro Suzuki 1 ; 1 Infectious disease, Hamamatsu university
school of medicine, Hamamatsu, Japan; 2 2nd department
of internal medicine, Hamamatsu university school of medicine,
Hamamatsu, Japan
Background and Aim: The mechanisms by which infection
with hepatitis C virus (HCV) modulates the process of liver
fibrosis still remains poorly defined. Increased expression of
pro-fibrogenic growth factors including transforming growth
factor-β (TGF-β) and extra cellular matrix proteins has been
correlated with the degree of fibrosis in the liver. The aim of
this study was to elucidate the mechanism of increased expression
of TGF-β mediated by HCV infection. Methods: Fibrogenic
responses caused by HCV (J6/JFH-1 isolate) infection was
assessed using co-culture system. Hepatocellular carcinoma
Huh7 cells infected with HCV were co-cultured with hepatic stellate
TWNT4 cells. TGF-β promoter activities were analyzed by
the luciferase reporter assay. Expression of mRNAs of fibrotic
markers was analyzed by real-time RT-PCR. Protein expression
was assessed by Western blotting and immunofluorescence.
Knockdown and knockout for targeted genes were performed
using siRNA and CRISPR-Cas9 systems, respectively. Results:
Expression of TGF-β1 and TGF-β2 was significantly higher
in HCV-infected cells than in non-infected cells; in particular,
gene expression and promoter activity of TGF-β2 were markedly
increased in cells with HCV infection or expression of the
viral polyproteins such as Core through NS2. Compared to
HCV-infected Huh7 alone or TWNT4 alone, co-culture of the
infected Huh7 and TWNT4 cells showed significantly higher
expression of collagen type1A1 (COL1A1). The upregulation
of COL1A1 was suppressed by TGF-β2 knockout in Huh7 cells.
From mutagenesis together with search for transcription factor
binding sites, two regions; previously-identified CRE and
temporal “CRE binding protein, hepatocyte-specific (CRE-
BH)-response” element (CREBHRE) within the proximal TGF-β2
promoter are important for its transcription regulation. Gel shift
assay showed CREBH binding to the regions. TGF-β2 promoter
activity was decreased by CREBH knockdown or knockout, and
was increased by expression of the active form of CREBH. ChIP
assay showed that binding of CREBH and ATF2 to TGF-β2 promoter
was increased when the HCV Core-NS2 was expressed,
under which precursor CREBH was processed into its active
form. Discussion: CREBH, an endoplasmic reticulum (ER)-localized,
liver-enriched transcriptional factor, is well-known to be
activated by ER-stress. Our findings suggest that the expression
of HCV proteins in the virus-infected cells potentially induces
ER-stress and CREBH activation. Increased expression of TGFβ2
by CREBH activation and its release from the infected cells
contributes to the induction of fibrogenic responses via paracrine
signaling to nearby hepatic stellate cells.
Disclosures:
The following authors have nothing to disclose: Takeshi Chida, Masahiko Ito,
Kazuhito Kawata, Yoshimasa Kobayashi, Tetsuro Suzuki
1394
Expression of ENTPD1/CD39 is protective in a mouse
model of biliary fibrosis
Linda Feldbrügge 1,3 , Shuji Mitsuhashi 1 , Eva Csizmadia 1 , Xiaofeng
Sun 1 , Moritz Schmelzle 2 , Simon C. Robson 1 ; 1 Gastroenterology,
BIDMC, Boston, MA; 2 Department for Surgery, Charité, Berlin,
Germany; 3 Department for Surgery, University Hospital Leipzig,
Leipzig, Germany
Introduction. Ecto-nucleoside triphosphate diphosphohydrolases
(ENTPD) comprise a family of cell surface located
transmembrane proteins that regulate purinergic signaling by
catalyzing extracellular nucleotides, such as ATP, ADP, to ultimately
generate adenosine. These mediators are important signaling
molecules in hepatic injury and inflammation. ENTPD1/
CD39 is expressed on endothelium, and sinusoidal immune
cells, and closely regulates liver regeneration by generating
AMP from ATP and ADP. ENTPD2/CD39L1 is expressed on
portal fibroblasts and perivascular cells and is a preferential
ecto-ATPase, generating ADP. The role of ENTPD-mediated
catalysis in modulating liver and biliary fibrosis is currently
unclear as both ATP and adenosine could promote fibrogenic
signals in portal myofibroblast and stellate cells and ADP is a
potent platelet agonist. Methods. C57BL6 wild type, ENTPD1
null mice and ENTPD2 null mice were subjected to bile duct
ligation (BDL). Blood and liver tissue were harvested at 2 and
4 weeks after BDL. Liver function, tissue injury and extent of
cholestasis were determined by serum liver function tests and
histopathology. To assess the extent of biliary fibrosis, we also
analyzed tissue hydroxyproline content and performed Masson’s
trichrome staining. Results. We observe a trend towards
decreased survival in the ENTPD1 null mice after BDL. ENTPD1
null mice further show significantly more weight loss, develop
more pronounced cholestasis, and more severe liver fibrosis
than wild type mice. No major profibrogenic effects are noted
in ENTPD2 null mice, as determined by hydroxyproline content
and morphometric analysis of peribiliary collagen deposition.
Conclusions. Purinergic signaling regulated by ENTPD1 limits
biliary fibrosis. In contrast, ENTPD2 seems to have less pronounced
effects. These opposing roles are most likely due to
differential catalytic function and localization of both enzymes
with ENTPD1 as the dominant, vascular endothelial ecto-enzyme.
Our findings further suggest a dominant role of ATP-mediated
purinergic receptor signal transduction during biliary
fibrogenesis.
Disclosures:
Moritz Schmelzle - Grant/Research Support: Novartis GmbH
Simon C. Robson - Grant/Research Support: Pfizer, NIH, Dainippon; Independent
Contractor: Biolegend, EMD Millipore, Mersana; Management Position:
eBioscience; Speaking and Teaching: ACP, Elsevier, ATC; Stock Shareholder:
Nanopharma, Puretech
The following authors have nothing to disclose: Linda Feldbrügge, Shuji Mitsuhashi,
Eva Csizmadia, Xiaofeng Sun