studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 899A
sesses anti-fibrogenic activity. A recent study showed a link
between Nuak2 and TGF-β, a major driver of hepatic fibrogenesis.
However, little information is available on the role
of Nuak2 in liver fibrosis, and in particular on HSC trans-differentiation.
Aims of this study was to elucidate the involvement
of Nuak2 in HSC transactivation, and to investigate the
possible interaction between Nuak2 and TGF-β signaling.
Methods: HSC were isolated from normal rat and human livers
and activated by culture on plastic. Knockdown of Nuak2 was
achieved by siRNA. Cell migration was evaluated in modified
Boyden Chambers. Protein expression and signaling pathways
were analyzed by Western blotting. Results: In fully activated
HSC, down-regulation of Nuak2 positively modulated cell
migration and induced changes in the expression of molecules
involved in cytoskeletal organization. Knockdown of Nuak2
increased α-SMA expression and phosphorylation of SMAD3,
in HSC exposed to TGFβ. Moreover, Nuak2 expression was
up-regulated in HSC following TGFβ treatment. Conclusions:
The AMPK related kinase, Nuak2, is modulated during the activation
process of HSC, regulates cytoskeletal organization and
cell motility and interacts with TGF-β in regulating the pro-fibrogenic
properties of HSC.
Disclosures:
Massimo Pinzani - Advisory Committees or Review Panels: Intercept Pharmaceutical,
Silence Therapeutic, Abbot; Consulting: UCB; Speaking and Teaching:
Gilead, BMS
Fabio Marra - Advisory Committees or Review Panels: Abbvie; Consulting: Bayer
Healthcare; Grant/Research Support: ViiV
The following authors have nothing to disclose: Cristina Tosti Guerra, Alessandra
Caligiuri, Angela Provenzano, Krista Rombouts
1411
The bile acid-phospholipid conjugate Ursodeoxycholyl
Lysophosphatidylethanolamide (UDCA-LPE) disturbs
pro-fibrogenic Integrin and TGFβ signaling
Jie Su, Hongying Gan-Schreier, Walee Chamulitrat, Wolfgang
Stremmel, Anita Pathil; Department of Internal Medicine IV, University
of Heidelberg, Heidelberg, Germany
BACKGROUND: Integrin receptors, which are involved in cellcell
and cell-matrix interaction emerge as crucial mediators of
TGFβ1 activation in liver fibrosis. Ursodeoxycholyl Lysophosphatidylethanolamide
(UDCA-LPE) is a synthetic bile acid-phospholipid
conjugate with hepatoprotective and anti-fibrogenic
functions in vitro and in vivo. In this study we aim to elucidate
signaling pathways, which mediate anti-fibrogenic action of
UDCA-LPE. RESULTS: In order to promote pro-fibrogenic signaling
upon extracellular matrix binding integrins recruit focal
adhesion kinase (FAK) and SRC kinase, which are phosphorylated
in response to integrin engagement. Incubation of CL48
liver cells with UDCA-LPE altered the localization of integrin α2,
α3, α5, αv, β1, β4, β5 and β6 as observed by immunofluorescence.
In contrast, incubation with the integrin blocking peptide
RGD reduced integrin protein levels, but did not change its
localization. After UDCA-LPE treatment integrins were transported
to the ER and the nuclear envelop, which led to a loss of
co-localization of integrins with SRC resulting in dephosphorylation
of FAK (Tyr 925 and Tyr 576/577) and SRC (Tyr416).
This translocalization of integrins was not induced by UDCA
and/or LPE treatment, but was exclusively achieved by UDCA-
LPE. To further dissect whether UDCA-LPE would mediate a
shift of integrins to certain membrane microdomains such as
lipid raft and non-raft regions lipid fractioning was performed.
Integrin α2, α3, α5, αv, β1, β4 and β6, but not SRC, was
significantly reduced in transitional fractions and increased in
lipid raft fractions. Taken together, UDCA-LPE disturbed integrin-FAK
signaling after short activation by modifying the localization
of integrins. UDCA-LPE further led to a shift of TGFβ1
receptor I and II to the ER and the nuclear envelope resulting
in dephosphorylation of Smad2/3. According to lipid fractionation
TGFβ1 receptor I and II were also reduced in transitional
fractions and increased in lipid raft fractions after UDCA-LPE
treatment. Analysis by HPLC-MS revealed that UDCA-LPE shows
an integrated fractionation of UDCA and LPE, suggesting that
the localization of UDCA-LPE depends on both UDCA and LPE
end. CONCLUSIONS: UDCA-LPE mediated translocation of
integrins and TGFβ1 receptors into lipid rafts leads to a loss of
colocalization with their down-stream signaling proteins SRC
and Smad2/3. By inhibiting crucial pro-fibrogenic signaling
pathways UDCA-LPE emerges as a promising experimental
drug-candidate for the treatment of liver fibrosis.
Disclosures:
The following authors have nothing to disclose: Jie Su, Hongying Gan-Schreier,
Walee Chamulitrat, Wolfgang Stremmel, Anita Pathil
1412
Curcumin blocks the loss of lipid droplets in activated
hepatic stellate cells by increasing perilipin 5 gene
expression in vitro
DingYu Zhang; Hepatology, Wuhan Medical Treatment Center,
Wuhan, China
BACKGROUND: Activation of hepatic stellate cells(HSCs) are
the major players during liver fibrogenesis, and featured by the
loss of intracellular lipid droplets(LD). Currently, accumulating
evidence showed that blockade of LD loss could inhibit HSC
activation including decreasing cell proliferation and extracellular
matrix production. Curcumin, a phytochemical from
turmeric, has been demonstrated to inhibit HSC activation and
protect the liver from fibrogenesis in vitro and in vivo. However,
whether curcumin affects intracellular LD formation in
HSCs and underlying mechanism remain largely undefined.
AIM AND HYPOTHESIS: The aims of this study are to evaluate
roles of curcumin in the formation of LD in HSC, and to further
explore the underlying mechanisms. It is hypothesized that curcumin
might eliminate the loss of LD in activated HSCs, rather
than hepatocytes, by increasing perilipin 5(plin5) gene expression
and inducing lipogenesis. RESULTS: Curcumin restored
intracellular LD formation, and increased triglyceride (TG) and
free fatty acid(FFA) levels as well as induced expression of
genes closely relevant to lipogenesis including SREBP-1c, fatty
acid synthase(FAS), PPAR-coactivator (PCA), in passaged mice
HSCs. In contrast, under the same condition, the LD formation
and lipid contents have been unaffected by curcumin in
mice hepatocytes. Furthermore, curcumin dose-dependently
increased plin5 gene expression which has been suppressed
in activated state in HSCs. Meanwhile, transduction with lentiplin5-YFP
in passaged HSCs had the similar effect as curcumin
treatment. On the contrary, in HSCs transfected with plin5-
siRNA prior to curcumin treatment, LD contents and lipid levels
were not changed significantly compared with the normal cells.
CONCLUSIONS: Our results demonstrate that curcumin restores
the LD formation in activated HSCs in vitro, which is carried
out by inducing lipid synthesis via increasing lipogenesis-related
gene expression. The process is mediated by activating
plin5 gene expression. Our results provide novel insight into
the mechanism of the role of curcumin in the inhibition of HSC
activation and hepatic fibrogenesis and potential therapeutic
strategies for treatment of hepatic fibrogenesis without increasing
lipotoxicity in hepatocytes.
Disclosures:
The following authors have nothing to disclose: DingYu Zhang