studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 897A
during fibrosis and cirrhosis, 48 were components of axon
guidance signaling pathways (P=4.65E-07, Enrichment=2.53),
which transcripts encoding ligands (slit2, Sema4D), membrane
receptors (Robo1 and 2, EphA and B, CXCR4, Plexin A4), and
downstream kinases (PAK1, 2,3 and 6, Gnai 1 and 2, Rock2,
and Mapk3). By immunoflurescence in immortalized human
and mouse hepatic stellate cell (HSC) lines LX-2 and JS1 in
vitro, and in the CCl4 induced fibrotic liver samples in vivo,
Robo2, a key receptor mediating axon guidance signaling,
was localized to the HSC surface. The amount and localization
of its protein expression was correlated well with fibrotic septa
in fibrotic livers. Conclusion: There is significant upregulation
and activation of axon guidance signaling pathway members
in liver during fibrogenesis. This signaling may have regulate
actin cytoskeleton, cell attraction and outgrowth of HSC. The
study also revealed RoBo2 as a novel HSC surface marker and
a potential therapeutic and diagnostic target of liver fibrosis.
Disclosures:
Scott L. Friedman - Advisory Committees or Review Panels: Pfizer Pharmaceutical;
Consulting: Conatus Pharm, Exalenz, Genfit, Exalenz Biosciences, Eli Lilly PHarmaceuticals,
Fibrogen, Boehringer Ingelheim, Nitto Corp., Immune Therapeutics,
Synageva, Roche/Genentech Pharmaceuticals, DeuteRx, Abbvie, Novartis,
RuiYi, Kinemed, Sanofi Aventis, Takeda Pharmaceuticals, Nimbus Therapeutics,
Bristol Myers Squibb, Astra Zeneca, Sandhill Medical Devices, Galmed, Northern
Biologics, Enanta Pharmaceuticals, Regado Bioscience, Raptor Pharmaceuticals,
Teva Pharmaceuticals, Zafgen Pharmaceuticals, Merck Pharmaceuticals,
Debio Pharmaceuticals; Grant/Research Support: Galectin Therapeutics, Tobira
Pharm; Stock Shareholder: Angion Biomedica, Intercept Pharma
The following authors have nothing to disclose: Jinsheng Guo, David Zhang,
Yujing Wu, Daisuke Hasegawa
1406
PDGFRα ubiquitination leads to its p62 mediated autophagic
degradation in a synectin dependent manner in
HSC
Haibin Yu, Mary Drinane, Usman Yaqoob, Vikas K. Verma, Thiago
de Assuncao, Sheng Cao, Vijay Shah; Gastroenterology Research
Unit, Mayo Clinic, Rochester, MN
Background/Aim: The platelet derived growth factor receptor
(PDGFR) signaling pathway is critical for hepatic stellate cell
(HSC) activation and liver fibrosis. While the critical role of
PDGFRβ is well studied, recent work suggests importance of
PDGFRα as well, making its regulation of interest. Synectin is a
scaffold protein that is a key regulator of endocytic trafficking
of multiple tyrosine kinases. Prior studies showed that knockdown
of synectin from HSC led to autophagic degradation
of PDGFRα. p62 is a ubiquitin-binding autophagy receptor
which selectively recognizes autophagic cargo and mediates
engulfment into autophagosomes in coordination with LC3B.
We hypothesized that synectin regulates selective autophagic
degradation of PDGFRα by recognizing specific ubiquitin sites
which in turn govern p62 binding with PDGFRα. Methods/
Results: Co-immunoprecipitation (co-IP) of PDGFRα and PDG-
FRβ was used to investigate p62 binding using HSC lysates.
p62 co-precipitated with PDGFRα, but not PDGFRβ. In response
to PDGF ligand, co-precipitation of PDGFRα with p62 was further
increased by 4.4 fold (p < 0.05). Synectin knockdown led
to a 2-fold increase in binding of p62 with PDGFRα (p < 0.05)
and increased co-localization of p62 and PDGFRα by confocal
microscopy. Sequence analysis of PDGFRα and PDGFRβ was
performed which identified two ubiquitin sites present within
α but not β subunits. Site directed mutagenesis of PDGFRβ
was performed to mutate the two corresponding residues to
lysine (Q613K and R979K) and generate a mutant PDGFRβ
construct (PDGFRβ- Q613K, R979K) that could be used to test
the hypothesis that these two lysine residues may be critical for
ubiquitin dependent PDGFR autophagic degradation. PDGFRβ-
Q613K, R979K underwent autophagic degradation, akin to
PDGFRα, while the wild-type PDGFRβ did not (p < 0.05). Conclusion:
The specificity of interaction of the autophagic receptor
p62 with PDGFR is conferred by specific ubiquitin sites and is
mediated by synectin.
Disclosures:
The following authors have nothing to disclose: Haibin Yu, Mary Drinane, Usman
Yaqoob, Vikas K. Verma, Thiago de Assuncao, Sheng Cao, Vijay Shah
1407
Reactive gamma-ketoaldehydes induce a proinflammatory
phenotype, oxidative stress, and activation of autophagy
in primary human hepatic stellate cells
Lisa Longato 1 , Fausto Andreola 1 , Sean S. Davies 2 , Jackson L. Roberts
2 , Giuseppe Fusai 1 , Massimo Pinzani 1 , Kevin Moore 1 , Krista
Rombouts 1 ; 1 Medicine, University College London, London, United
Kingdom; 2 Vanderbilt University, Nashville, TN
Background and aims. Products of oxidative stress such as
4-hydroxynonenal are key activators of hepatic stellate cells
(HSCs). γ-Ketoaldehydes (γ-KAs), including levuglandins and
their isomers, are a family of acyclic aldehydes, which are
formed during oxidation of arachidonic acid, or as a by-product
of the cyclooxygenase pathway. γ-KAs are highly reactive
and form protein adducts and cross-links at a rate > 100-fold
compared to 4-HNE. Increased serum antibodies against proteins
cross-linked to γ-KAs are present in patients with alcoholic
liver disease. Since the contribution of γ-KAs to liver injury has
not been studied, we sought to investigate the effects of γ-KA
levuglandin E 2
(LGE 2
) on the cell biology of primary human
HSCs. Methods. Primary human HSCs were exposed to LGE 2
(0.5 pM- 5 mM) for up to 48 hours and analyzed for proliferation,
cytotoxicity (lactate dehydrogenase, MTS, and Neutral
Red assays), RNA/protein expression, reactive oxygen
species (ROS) production, and ultrastructure. Results. Exposure
to a 5 μM dose of LGE 2
was profoundly cytotoxic and
resulted in apoptotic cell death, as indicated by LDH leakage,
reduced MTS and Neutral Red incorporation, increased levels
of cleaved PARP, CHOP, and JNK phosphorylation. Lower,
non-cytotoxic doses (50 nM-500 nM) of LGE 2
induced selected
indices of HSCs activation, such as increased expression of
a-smooth muscle actin (α-SMA), and activation of signaling
pathways (ERK1/2). In contrast, LGE 2
had no effect on DNA
synthesis or pro-fibrogenic markers, particularly collagen type
I, and lysyl oxidase (LOX). In contrast, HSCs exposed to LGE 2
displayed a marked increase in expression for various cytokines/chemokines
including interleukin-8, -6, -1β, and MCP-1.
This was accompanied by increased secretion of bioactive
IL1β/IL-18, as measured by HEK-Blue reporter cells. Pre-incubation
of cells with the either chemicals inhibitors of JNK
(SP600125), or NFkB (PDTC), but not p38MAPK (SB203580),
partially reduced these increases in cytokines and chemokines.
Ultrastructural analyses revealed that LGE 2
-treated HSCs had
prominent formation of autophagic vescicles, suggesting ongoing
autophagy, which was confirmed by immunofluorescence
for LC3B, as well as by accumulation of LC3II. Lastly, shortterm
exposure to LGE 2
promoted a dose-dependent formation
of ROS in the cells, as measured by the carboxy-H 2
DCFDA
fluorescent probe. Conclusions. γ-KAs represent a newly identified
class of activators of HSCs in vitro, which are biologically
active at concentrations as low as 50 nM, and are particularly
effective at promoting a pro-inflammatory response, as well as
oxidative stress, and autophagy.
Disclosures:
Massimo Pinzani - Advisory Committees or Review Panels: Intercept Pharmaceutical,
Silence Therapeutic, Abbot; Consulting: UCB; Speaking and Teaching:
Gilead, BMS