CHONDROCYTE MATURATION AND SKELETAL GROWTH ARE IMPAIRED IN COL2A1-ICAT TRANSGENIC
MICE
Chen, M.; Zhu, M.; Boyce, B.F.; O’Keefe, R.J.; and +Chen, D.
+Center for Musculoskeletal Research, University of Rochester, School of Medicine and Dentistry, Rochester, NY
di_chen@urmc.rochester.edu
Introduction: Wnt/β-catenin signaling provides various points for
antagonist activity. Extracellular inhibitors include the secreted Frizzledrelated proteins (sFRP), Wnt inhibitory factor-1 (WIF), and Cerebus.
Another class of inhibitors involves the Dickkopf (Dkk) family, which
are membrane-associated proteins that bind Wnt receptor complexes to
inhibit signaling. While these factors have been intensively investigated,
intracellular inhibitors, including Inhibitor of β-catenin and TCF (ICAT)
have received less attention.
ICAT is an 81 amino acid protein that binds β-catenin at its
Armidillo repeat region and thus acts as a competitive inhibitor of
TCF/LEF binding. Expression and function of ICAT have not been
studied in cartilage or bone. Here we present findings that define ICAT
as a critical regulator in chondrocytes. We show that ICAT i) is
expressed in chondrocytes, ii) is regulated by Wnt 3a and TGF-β, and
iii) inhibits β-catenin signaling in chondrocytes. Finally we have
established several lines of a Col2a1-Flag-ICAT transgenic mouse and
demonstrate delayed chondrocyte maturation in vivo.
Materials and Methods: Mouse strains. Topgal transgenic mice were
obtained form the Jackson Laboratory. Flag-ICAT cDNA was cloned
into PKN185 vector and the transgene containing 1.0kb Col2a1
promoter and Flag-ICAT followed by the Col2a1 enhancer was released
by NdeI and HindIII digestion. The PCR genotyping was performed
using
primers
5'-atggactacaaggacgacgatgac-3'
and
5'gtctggatccccgcggccgc-3'. β-gal staining. Mouse embryos were fixed in
2% paraformaldehyde and 0.02% glutaraldehyde in PBS for 1h at room
temperature and washed twice in PBS. The embryos were incubated in
0.1% X-gal, 2 mM MgCl2, 5 mM EGTA, 0.02% Nonidet P-40, 5 mM
K3Fe(CN)6, and 5 mM K4Fe(CN)6 at 30°C overnight, then postfixed in
4% paraformaldehyde. Histology. Samples were formalin-fixed, paraffin
embedded, and sections stained with Hematoxylin/Eosin. Isolation and
culture of primary chondrocytes. Primary chondrocytes were obtained
from 3-day-old mice by enzymatic digestion of sternum, and cultured in
DMEM with 10% FBS and 50μg/ml ascorbic acid. RNA isolation and
real time RT-PCR. RNA was extracted using Trizol and cDNA
synthesized with Iscript Kit (BIORAD). Real time PCR was performed
using primers specific for murine ICAT.
Results:
β-catenin signaling is present in chondrocytes and is inhibited by ICAT.
To determine if β-catenin signaling is active in chondrocytes, we
performed lacZ staining in embryos of Topgal transgenic mice in which
expression of lacZ transgene is driven by the β-catenin-responsive
element, 3xTCF. Intensive β-galactosidase staining was present in
proliferating and hypertrophic chondrocytes in 14.5 and 16.5 dpc
embryos of Topgal transgenic mice, suggesting that β-catenin signaling
is active in chondrocytes during endochondral bone formation. We
investigated the effect of ICAT on chondrocyte proliferation and
differentiation. Transfection of ICAT expression plasmid into
chondrocyte cell line C5.18 and TMC-23 cells inhibited Topflash
reporter activity induced by β-catenin.
ICAT is expressed in chondrocytes and is differentially regulated by
Wnt3a and TGF-β.
ICAT expression and regulation were examined in primary murine
chondrocytes treated with Wnt3a (100ng/ml), BMP-2 (100ng/ml), and
TGF-β (5ng/ml) for 48 hours. BMP-2 had no effect, while Wnt3a
increased ICAT mRNA expression (30%), suggesting a compensatory
mechanism to down-regulate β-catenin signaling. In contrast, TGF-β
inhibited ICAT expression (35%). Since related experiments
demonstrated that TGF-β increased cellular β-catenin levels and induced
Topflash promoter activity, inhibition of ICAT may be a mechanism
involved in induction of β-catenin signaling by TGF-β. These findings
establish that ICAT is expressed, modulates β-catenin signaling, and is
regulated by factors that control chondrocyte maturation.
To determine the role of ICAT in chondrocyte maturation in vivo,
we generated a transgene using Flag-tagged ICAT cDNA driven by the
Col2a1 promoter (1.0 kb). Expression and biological activity were
established in TMC-23 chondrocytes. Col2a1-ICAT inhibited basal and
β-catenin stimulated Topflash reporter activity, and the effect was similar
to the level of inhibition caused by pcDNA3-ICAT.
Pro-nuclear injections resulted in the generation of seven Col2a1ICAT founder mice and the establishment of two independent transgenic
lines (Line 1 and 5). Expression of Flag-ICAT protein was confirmed by
Western blot and by immunostaining using anti-Flag and anti-ICAT
antibodies. Expression of Flag-ICAT was observed in chondrocytes
from transgenic mice, but was absent in wild type mice. The majority of
Col2a1-ICAT transgenic mice survive into adulthood. Radiographic and
histological evaluation show reduced growth and morphological
abnormalities of the growth plate and epiphysis. Col2a1-ICAT
transgenic mice are approximately 30-50% smaller than wild type
littermates and histological analysis demonstrates delayed chondrocyte
differentiation. Sections obtained from the distal femur and proximal
tibia of 2-week old Col2a1-ICAT transgenic mice shows that immature
chondrocytes continue to populate the epiphysis, in comparison to wild
type littermates that have hypertrophic cartilage and formation of
secondary centers of ossification. In addition, the hypertrophic region of
the growth plate is substantially shorter in Col2a1-ICAT transgenic
mice.
Primary chondrocytes from Col2a1-ICAT transgenic mice have reduced
β-catenin signaling and a less differentiated phenotype.
Primary sternal chondrocytes isolated from 3-day-old transgenic
mice had approximately a 70% reduction in both basal and stimulated
Topflash reporter activity compared to transfected wild type cells. The
maturation marker, alkaline phosphatase was reduced 50% in
chondrocytes derived from Col2a1-ICAT transgenic mice consistent
with a stimulatory role for β-catenin on chondrocyte maturation. Finally,
while TGF-β resulted in a 2.5-fold stimulation of col2 expression in wild
type chondrocytes, no effect was observed in transgenic mice.
Discussion: The present studies establish that ICAT is an important
regulator of endochondral bone formation. ICAT is expressed by
chondrocytes, is regulated by Wnt3a and TGF-β, and modulates βcatenin signaling. Furthermore, in vivo findings show that overexpression of ICAT results in profound morphological abnormalities of
cartilage and impaired long bone growth. In vitro studies using cells
obtained from Col2a1-ICAT transgenic mice confirm that the transgene
is active and inhibits β-catenin signaling. To our knowledge, the Col2a1Flag-ICAT transgenic mouse is the first in vivo model in which either
gain or loss of β-catenin function in cartilage has survived the post natal
period. Thus, the findings for the first time demonstrate that β-catenin
signaling has an essential role in post-natal growth and development.
Absence of β-catenin signaling due to ICAT over-expression was
associated with a marked delay in chondrocyte maturation in growing
mice. Taking together, the findings demonstrate an essential role for
ICAT as an intracellular regulator of β-catenin signaling and
chondrocyte maturation.
W ild-type (tibia)
10x
HC
HC
Generation of Col2a1-ICAT transgenic mice.
52nd Annual Meeting of the Orthopaedic Research Society
Paper No: 0379
Col2a1-ICAT transgenic (tibia)
10x
Figure 1. Inhibition of epiphyseal bone form ation and
chondrocyte m aturation in C ol2a1-ICAT transgenic
m ice. Histological analysis (Alcian blue/H&E staining) of
a 2-week-old Col2a1-ICAT transgenic mouse and its wt
littermate showed that epiphyseal bone formation (black
arrow) and chondrocyte maturation (black bar) are
significantly delayed in Col2a1-IC AT transgenic mouse.
HC: hypertrophic chondrocytes