96. Jahrestagung der Deutschen Gesellschaft für Pathologie e. V ...

SO-075
A novel, dual role of CCN3 in experimental glomerulonephritis:
pro-angiogenic and anti-mesangioproliferative effects
P . Boor 1 , C . van Roeyen 1 , E . Borkham-Kamphorst 2 , S . Rong 1 , U . Kunter 1 ,
I .V . Martin 1 , A . Kaitovic 1 , S . Fleckenstein 1 , B . Perbal 3 , R . Weiskirchen 2 , T . Ostendorf
1 , J . Floege 1
1 RWTH Aachen University, Aachen, 2 RWTH Aachen University, Inst . of
Clinical Chemistry and Pathobiochemistry, Aachen, 3 R&D L’Oréal, Clark, NJ,
United States
Aims. In contrast to factors promoting mesangial cell proliferation, little
is known about their endogenous inhibitors. During experimental mesangioproliferative
nephritis, glomerular CCN3 (also known as NOV or
nephroblastoma overexpressed gene) expression is reduced prior to the
proliferative phase and overexpressed in glomeruli and serum when mesangial
cell proliferation subsides.
Methods. To further elucidate its role in mesangioproliferative glomerulonephritis,
CCN3 was systemically overexpressed by muscle electroporation
in healthy or nephritic rats. This increased CCN3 serum concentrations
more than 3-fold for up to 56 days.
Results. At day 5 after disease induction, CCN3-transfected rats exhibited
an increase in glomerular endothelial area and in glomerular mRNA
levels of the pro-angiogenic factors VEGF and PDGF-C. In the mesangioproliferative
phase (day 7), CCN3 overexpression decreased mesangial
cell proliferation including expression of α-smooth muscle actin and
matrix accumulation of fibronectin and type IV collagen. In progressive
nephritis (day 56), overexpression of CCN3 resulted in decreased albuminuria,
glomerulosclerosis and reduced cortical collagen type I accumulation.
In healthy rat kidneys, overexpression of CCN3 induced no
morphological changes but regulated glomerular gene transcripts (reduced
transcription of PDGF-B, PDGF-D, PDGFR-β and fibronectin and
increased PDGFR-β and PDGF-C mRNA).
Conclusions. The above data identify a dual role of CCN3 in experimental
glomerulonephritis with pro-angiogenic and anti-mesangioproliferative
effects. Manipulation of CCN3 may represent a novel approach to help
repair glomerular endothelial damage and mesangioproliferative changes.
Deutsch-Chinesisches Symposium
Colorectal Carcinoma
SG-129
ACSL5, a modifier of WNT activity
C . Klaus1 , U . Schneider1 , R . Knuechel1 , N . Gaßler1 1RWTH Aachen University, Institute of Pathology, Aachen
Aims. The mitochondrial localized Acyl-CoA synthetase 5 (ACSL5) converts
free long-chain fatty acids into fatty acyl-CoA esters, and thereby
plays a key role in lipid biosynthesis and fatty acid degradation. In particular,
ACSL5 has been recently identified to be involved in apoptotic
cell death of senescent enterocytes along the intestinal crypt-villus axis
and to interact with mitochondrial proteins. The aim of this study was
to investigate ACSL5-dependent effects on intestinal signalling pathways
that coordinate proliferation and/or differentiation of enterocytes, particularly
the Wnt pathway.
Methods. Wnt signalling was analysed in an ACSL5 overexpressing cell
culture model using luciferase assays, immunohistochemistry, qRT-PCR
and Western blot. The findings were substantiated with expression studies
in human colon carcinomas and in a Wnt-associated mouse model.
Results. ACSL5 transgenic intestinal-derived cells displayed a strong
susceptibility to pro-apoptotic stimuli. This phenomenon was accom-
panied by caspase-3 activation and significant down-regulation of Wnt
signalling activity. In particular, Wnt pathway-associated mitochondrial
localized molecules were identified as interaction partners of ACSL5 activity.
Conclusions. Molecular mechanisms underlying ACSL5-dependent
apoptosis susceptibility of enterocytes are probably bivalent including
pro-apoptotic and anti-proliferative activities.
SG-130
Regulation of differential WNT activity in colorectal cancer
D . Horst1 , J . Chen2 , T . Kirchner1 , R . Shivdasani2 1Ludwig-Maximilians-Universität München, Pathologisches Institut,
München, 2Harvard Medical School, Dana-Farber Cancer Institute, Boston,
United States
Aims. Most colorectal cancers (CRCs) express the WNT-effector protein
β-catenin in a heterogeneous pattern. Strong nuclear expression is
often confined to a small fraction of tumor cells at the tumor’s leading
edge. Recent data suggest a role for Mitogen Activated Protein Kinase
(MAPK) signaling in nuclear accumulation of beta-catenin. We therefore
investigated if MAPK activity regulates overtly differential WNT
activity in CRC cell subpopulations.
Methods. We used gene expression profiling, and immunohistochemistry
to assess interdependence of MAPK and WNT pathway activity in
CRC. Lentivirus- and drug-based pathway modification in CRC xenograft
tumors of primary human colon cancers and colon cancer cell lines
was used to study the effect of MAPK activation or repression on differential
WNT activity.
Results. CRC cells with high WNT activity showed coincident overexpression
of MAPK target genes and high levels of phospho-ERK, indicating
active MAPK signaling. Forced MAPK activation by lentiviral
expression of constitutively active KRAS enhanced WNT pathway activity
in vivo in CRC xenograft tumors, whereas drug based inhibition of
EGFR signaling attenuated it.
Conclusions. Although CRC is characterized by mutational activation of
the WNT pathway, MAPK signaling influences intratumoral β-catenin
heterogeneity, revealing a mechanism for external stimuli to modulate
pathway activity. Because MAPK signaling does not merely coincide
with nuclear β-catenin but also regulates it, this may account for the high
frequency of KRAS mutations in CRC.
SG-132
IGFBP7 and WNT signaling pathway in tumor stroma interactions
C . Rao1 , J . Xu1 , M . Liu1 , H . Deng1 1Department of Pathology, School of Medicine, Zhejiang, China
Aims. To find out the mechanism of the up-regulation of IGFBP7and the
biological changes in fibroblasts during the interactions with colorectal
cancer cells.
Fibroblasts (HELFs) were cultured in colorectal cancer cells conditioned
media (SW620-CM), treated by TGF-β 1, TGF-β1 receptor antagonist
(SB431542), TGF-β1 specific antibody (AF), Wnt signaling pathway
agonist (LiCl) and inhibitor (DKK-1) respectively. Q-PCR, Western Blot,
ELISA, Immunofluorescence microscopy and flow cytometry were used
to detect the expression of related targeted genes and proteins of TGF-β
and Wnt signaling pathways.
HELFs cultured in SW620-CM were activated with abundant expression
of α-SMA and showed strong proliferation and weak apoptosis and senescence.
The expression of IGFBP7 of HELFs was up-regulated in timedependent
and dose-dependent manners when cultured with SW620-
CM, while TGF-β signaling were activated as Smad2, P-Smad2 and
TGF-βRΠ were up-regulated in HELFs. These effects could be strengthened
by TGF-β1 and inhibited by SB431542 or AF. During the interactions,
the downstream genes of Wnt signaling pathway such as c-myc, cyclinD1
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