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Results: Ubr5mt mice demonstrated (i) progressive ectopic chondro-
genesis, calcification and ossification of various tissues, including the
knee synovium, Achilles tendon and retinaculum and (ii) progressive
and spontaneous articular cartilage degradation resulting in exposed
subchondral bone at six months. In agreement with the current dogma,
Ubr5mt tissue exhibited increased Hedgehog signalling. However,
genetic or pharmacological inhibition of Hedgehog signalling in a
Ubr5mt background caused a dramatic enhancement, rather than the
expected reduction, of articular cartilage damage and heterotopic
ossification. 12-week-old Ubr5mt and Smo-deficient animals exhibited
large ectopic bone masses around the ankle joint, ectopic cartilage
within the knee synovium and advanced articular cartilage degradation.
Experiments in juvenile animals also revealed that post-natal loss of
Ubr5 function was necessary and sufficient to promote heterotopic
ossification. Furthermore, application of cyclopamine to juveniles also
revealed a post-natal role for Smoothened-mediated Hedgehog sig-
nalling in regulating skeletal.
Conclusions: These data reveal a previously unknown role for UBR5 as
both a regulator of skeletal tissue homeostasis and regulator of
Hedgehog signalling. While our results support an important role for
Hedgehog signalling in skeletal homeostasis, it conflicts with the cur-
rent dogma of Hedgehog signalling promoting articular cartilage
damage and heterotopic ossification. This work highlights an important
new player in chondrocyte biology whose function, or associated
functions, could be affected in human disease and harnessed for bio-
medical applications.
168
UROCORTIN EXPRESSION IN CARTILAGE IS ATTENUATED IN A NON-
INVASIVE MOUSE MODEL OF POST TRAUMATIC OSTEOARTHRITIS
F. Ter Heegde y, R.C. Jones z, T.R. Jackson z, C. De Pascale x, I.C. Locke x,
T.N. Board k, S.M. Richardson z, P.A. Townsend z,K.M. Lawrence z.
y
Royal
Vet. Coll., London, United Kingdom;
z
Univ. of Manchester, Manchester,
United Kingdom;
x
Univ. of Westminster, London, United Kingdom;
k
Wrightington Hosp., Wigan, United Kingdom
Purpose: Traumatic injury to the joint commonly develops into an
accelerated form of osteoarthritis. This post traumatic osteoarthritis
(PTO) is frequently associated with sports injury and represents a sig-
nificant proportion of patients presenting with osteoarthritis. Currently,
there are no treatments other than analgesics and joint replacement
surgery. The development of PTO, although multifactorial, is closely
associated with chondrocyte cell death. We have recently found that the
peptide Urocortin (Ucn1), its receptor (CRF-R1) and regulatory binding
protein (CRF-BP) are expressed by chondrocytes, thus constituting an
intact and functional Ucn1 signalling system. Furthermore, in a cartilage
explant model of acute impact injury, we found that Ucn1 potently
protects chondrocytes from death when applied both pre- and post-
impact. These finding suggests a potential role for Ucn1 in preventing
cartilage tissue damage in PTO and as a novel therapeutic. Studies
investigating the effects of mechanical loading on articular cartilage
have been performed in vitro or with ex vivo cartilage explants. How-
ever, these are not ideal as crucial interactions between cells and their
environment are lost, and reliable pathology may be compromised by
dissection and culture. The most common in vivo models that simulate
mechanical injury involve surgical joint destabilisation; these are
severely disadvantaged by the need for surgery, increasing infection
risk, trauma, and effects on joint cell metabolism. There is, therefore, a
need for a nonsurgical model allowing for adjustment of specific
components of the applied load, including its magnitude, frequency,
and loading cycle shape, which can reflect the variable loads encoun-
tered throughout life and importantly display a developmental path-
ology similar to PTO in humans.
Methods: Here we used a non-invasive controlled axial loading of the
mouse tibia model, enabling loading through normal points of articu-
lation and precisely controlled mechanical loading regimens. In order to
induce osteoarthritis, the right knee of 12 week old male C57/bl6 mice
was loaded at 9N (pathological load). A single loading episode consisted
of 40 loading cycles with a trapezoidal wave form in which 9N was
applied for 0.05sec with a rise and fall time of 0.025sec followed by a
9.9sec rest time at a holding load of 2N. This loading episode was
repeated 3 times per week for two weeks after which animals were kept
for 6 more weeks with no loading before sacrifice (Schematic1). The
control group used consisted of mice receiving a 2N holding load
without any applied load. At week 8, knee joints exposed to both loads
were fixed, decalcified, embedded in paraffin and sectioned coronally at
6
m
m. These were then subjected to immunohistochemistry using
antibodies specific for Ucn1 itself, CRF-R1 and CRF-BP, and visualised
using an HRP conjugated secondary antibody.
Results: It has been previously demonstrated that the loading regimen
presented in Schematic1 causes significant development of PTO when
compared to a holding load alone. We found positive immunostaining
in cartilage tissue with all three antibodies for the Ucn system mem-
bers. Expression levels of CRF-R1 and CRF-BP did not alter with load
severity. However, we found that expression of Ucn1 itself was mark-
edly reduced in the cartilage of mice exposed to a 9N pathological load
when compared to the holding load of 2N.
Conclusions: We have found expression of Ucn system members in
articular cartilage at the protein level in vivo, including the ligand itself,
its receptor and regulatory binding protein, suggesting an intact sig-
nalling pathway and regulatory system.There was a profound decrease
in Ucn1 protein expression in the cartilage of joints exposed to a loading
regimen which develops into PTO. This finding suggests a post-injury
loss of Ucn1 signalling, caused by a decrease in Ucn1 expression, which
may be responsible for the initiation or progression of PTO.
169
TRANSIENT RECEPTOR POTENTIAL CATION CHANNEL (TRPC6) AS A
REGULATOR OF CXCR2-MEDIATED ARTICULAR CARTILAGE
HOMEOSTASIS
J. Sherwood y, M. Sambale y, J. Bertrand z, F. Dell'Acciox,T.Papy.
y
Univ.
Hosp. Muenster, Muenster, Germany;
z
Otto-von-Guericke Univ. of
Magdeburg, Magdeburg, Germany;
x
Queen Mary Univ. of London,
London, United Kingdom
Purpose: CXCR2 signalling is known to be required for maintenance of
articular cartilage homeostasis during conditions of physiological
challenge, indicated by increased cartilage degradation in CXCR2
-/-
mice
following destabilization of the medial meniscus. The transient receptor
potential channel 6 (TRPC6) mechanosensitive ion channel has pre-
viously been demonstrated as a specific mediator of CXCR2 driven cell
migration. Here, we aim to investigate the contribution of TRPC6
channel activity to CXCR2-mediated control of cartilage homeostasis.
Methods: 8 week old male wild type (WT) and TRPC6
-/-
mice were
subjected to a treadmill running model of induced osteoarthritis, run-
ning for 40 minutes per day, 5 days per week for 6 weeks. Knee joints
were analysed post-mortem by microCT, histology and OARSI score. W T
and TRPC6
-/-
chondrocyte TRPC channel and phenotypic gene and
protein expression was assessed using real time RT-PCR, Western blot
and immunohistochemistry. WT and TRPC6
-/-
chondrocyte pellet cul-
tures were analysed using Alcian blue staining and immunohis-
tochemistry. Murine CXCL6 was used to activate CXCR2, whilst hyp9, a
stabilized derivative of the TRPC6-selective activator hyperforin, was
used to specifically activate TRPC6. Intracellular calcium mobilization
was measured using a fluo-4 calcium influx assay.
Results: TRPC6
-/-
mice subjected to the treadmill running model
developed a more severe OA-like phenotype than WT controls. TRPC6
-/-
chondrocytes expressed lower levels of the chondrocyte differentiation
markers SOX9 and type II collagen in comparison to WT controls, which
was associated with lower levels of AKT phosphorylation. TRPC6
-/-
chondrocyte cultures produced less sulfated proteoglycans in compar-
ison to WT. In vitro activation of TRPC6 using hyp9 led to increased AKT
phosphorylation and resulted in a significant increase in SOX9 and type
II collagen mRNA expression, together with a decrease in the expression
of type X collagen. CXCL6 treatment of WT chondrocytes resulted in
increased intracellular calcium mobilization, however this response
was not observed in CXCL6 treated TRPC6
-/-
cells. CXCL6 treatment led
to an increase in pAKT levels in WT but not in TRPC6
-/-
chondrocytes,
whereas a non-specific increase in intracellular calcium levels achieved
using ionomycin led to AKT phosphorylation in both WT and TRPC6
-/-
cells.
Abstracts / Osteoarthritis and Cartilage 26 (2018) S60eS474S96