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First published online 23 April 2008
doi: 10.1242/dev.018044
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1 Genetic Disease Research Branch, National Human Genome Research Institute, 49
Convent Drive, MSC 4472, Bethesda, MD 20892, USA.
2 Endocrine Unit, Massachusetts General Hospital and Harvard Medical School,
Boston, MA 02114, USA.
3 Cardiovascular Research Institute, University of California, San Francisco, CA
94143, USA.
4 Laboratory of Pathology, National Cancer Institute, 9000 Rockville Pike,
Bethesda, MD 20892-1500, USA.
* Author for correspondence (e-mail: yingzi{at}mail.nih.gov)
Accepted 31 March 2008
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SUMMARY |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Key words: Ihh, Patched 1, Cartilage, PTHrP, Chondrocyte hypertrophy
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Karsenty and Wagner, 2002;
Kronenberg, 2003;
Zelzer and Olsen, 2003).
Furthermore, the proper regulation of chondrocyte hypertrophy is also
necessary for maintaining the cartilage lining synovial joint surfaces, as
abnormal chondrocyte hypertrophy in articular cartilage is associated with a
common cartilage degenerative disease: osteoarthritis
(Poole, 1999).
Indian hedgehog (Ihh) is a member of the hedgehog (Hh) family that plays
crucial roles in regulating many developmental processes. Ihh is one of the
key signaling molecules controlling both chondrocyte hypertrophy and bone
formation in the developing skeletal system. Ihh is expressed in
prehypertrophic chondrocytes and it regulates the onset of hypertrophic
differentiation by forming a negative feedback loop with parathyroid hormone
related protein (PTHrP; Pthlh - Mouse Genome Informatics)
(St-Jacques et al., 1999;
Vortkamp et al., 1996). Ihh
activates the expression of PTHrP in the periarticular cells and the
articular chondrocytes, and PTHrP signals through its receptor Pthr1 to
inhibit chondrocyte hypertrophy and to suppress further Ihh
expression by keeping chondrocytes in a proliferating state
(Lanske et al., 1996;
St-Jacques et al., 1999). Ihh
signaling is also required for chondrocyte proliferation and osteoblast
differentiation independently of PTHrP signaling
(Karp et al., 2000;
Long et al., 2004;
St-Jacques et al., 1999).
However, it remains to be elucidated whether Ihh signaling regulates
chondrocyte hypertrophy exclusively through PTHrP, as Ihh signals
directly to proliferating and prehypertrophic chondrocytes. In the
Ihh-/- embryo, PTHrP is not expressed and yet
chondrocyte hypertrophy is delayed at E14.5, although it is accelerated later
(St-Jacques et al., 1999). A
recent study demonstrates that Ihh promotes the transition from
periarticular chondocytes to proliferating chondrocytes and regulates columnar
cell mass independently of PTHrP signaling
(Kobayashi et al., 2005b). In
addition, Gli3, a downstream component of the hedgehog signaling pathway, acts
as a repressor to inhibit the transition of periarticular chondrocytes into
columnar chondrocytes by a PTHrP-independent mechanism
(Hilton et al., 2005;
Koziel et al., 2005). As
chondrocyte proliferation, which directly affects hypertrophy, was altered
when Hh signaling was manipulated in these studies, the
PTHrP-independent role of Ihh signaling in regulating chondrocyte
hypertrophy was not revealed.
Here, we have tested the role of Ihh in regulating chondrocyte
hypertrophy independently of PTHrP, by inactivating or activating Ihh
signaling cell autonomously in the developing cartilage in the
PTHrP-/- mouse embryo (in which cell proliferation is
greatly reduced). All Hh family members signal through two multipass
transmembrane proteins, Patched 1 (Ptch1) and Smoothened (Smo). In the absence
of Hh ligand, Ptch1 suppresses the activity of Smo. Upon Hh binding to Ptch1,
the inhibitory effect on Smo is relieved and Smo transduces the Hh signal to
downstream signaling components, which leads to the activation of Hh
downstream target gene expression, including Hip1 (Hhip -
Mouse Genome Informatics), Gli1 and Ptch1 (reviewed by
Huangfu and Anderson, 2006;
Lum and Beachy, 2004).
Therefore, when Smo is removed, Hh signaling is inactivated, whereas
removal of Ptch1 leads to the activation of Hh signaling
cell-autonomously. Here, we have investigated the PTHrP-independent
function of Ihh signaling in chondrocyte hypertrophy by genetically altering
the expression or function of Ihh, Ptch1 and Smo in
PTHrP-/- mouse embryos. Our results reveal a novel role of
Ihh signaling in promoting chondrocyte hypertrophy independently of
PTHrP, which is important in postnatal cartilage development,
although it is masked by the dominant effect of PTHrP-dependent Ihh
signaling in inhibiting chondrocyte hypertrophy during embryonic development.
We also found that canonical Wnt and Bmp signaling may mediate the role of Ihh
signaling in promoting chondrocyte hypertrophy.
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MATERIALS AND METHODS
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DISCUSSION
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).
Histology, in situ hybridization and immunohistochemistry
Embryos were fixed in 4% paraformaldehyde at 4°C overnight. Fixed
samples were embedded in paraffin and sectioned at 5 µm thickness.
Histological analysis, immunohistochemistry and radioactive 35S RNA
in situ hybridization were performed as described
(Yang et al., 2003). Primary
antibodies (anti-phospho-Smad1 goat polyclonal IgG (Santa Cruz) at 1:100 and
anti-Col2a1 goat polyclonal IgG (Santa Cruz) at 1:200 were used for
immunohistochemistry, and signals were detected using the ABC kits (Vector
Laboratories) and DAB (Sigma). Probes for in situ hybridization have been
described previously: Col10a1, Col2a1 and Ihh
(Mak et al., 2006); the
Lef1 probe was ordered from ATCC.
Organ cultures of embryonic limb explants
Forelimbs of mouse embryos were dissected free of skin and muscles at E14.5
and cultured for 1.5 days in BGJ-B medium (Invitrogen) with
Antibiotic/Antimycotic (Invitrogen) and 0.1% BSA in organ culture dishes under
humidified conditions. Cultures were supplemented with 2.5 µg/ml
recombinant murine Shh-N (R&D Systems) or 10 µM cyclopamine (BIOMOL).
Limb explants were then fixed with 4% paraformaldehyde overnight at 4°C
and embedded in paraffin for sectioning.
Preparation of primary chondrocytes
Ventral parts of the rib cages of 0- to 3-day-old wild-type pups were
eviscerated of skin and muscles and incubated with 2 mg/ml pronase (Roche) for
30 minutes at 37°C. The samples were then incubated with 3 mg/ml
collagenase D (Roche) in DMEM (Gibco) at 37°C for 1.5 hours until all soft
tissues had detached from the cartilage. The cartilage was washed with PBS
several times and separated from soft tissues by sedimentation. The cartilage
was then digested with collagenase for 4 to 5 hours in a Petri dish.
Undigested bony parts were discarded and chondrocytes were collected by
centrifugation and washed twice with PBS. The primary chondrocytes were
cultured in low-serum medium (DMEM, 0.1% FBS) for dual luciferase assays.
Dual luciferase assay
Primary chondrocytes were transfected with the Topflash reporter
plasmid using the Human Chondrocyte Nucleofector Kit (Amaxa), following the
manufacturer's protocol. After nucleofection, 0.5x106 cells
were seeded in 12-well plate and left to recover for 4-5 hours. Primary
chondrocytes were then serum starved overnight before adding 2.5 µg/ml
recombinant Shh (R&D Systems) was culturing for 24 hours. Cells were
harvested and subjected to luciferase activity measurement using the Dual
Luciferase Reporter Assay Kit (Promega), according to the manufacturer's
instructions.
Quantitative RT-PCR
Quantitative PCR was performed to measure the relative expression levels of
the Wnt genes and the Wnt target genes Axin2 and Lef1 (see
Table 1), using the Platinum
SYBR Green Kit (Invitrogen). Samples were normalized to Gapdh
expression.
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DISCUSSION
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).
Chondrocyte hypertrophy was analyzed by histological staining and in situ
hybridization with probes of Ihh and Col10a1, markers for
prehypertrophic and hypertrophic chondrocytes, respectively. The distance from
the articular end to the prehypertrophic or hypertrophic chondrocytes in the
developing cartilage reflects how fast chondrocytes undergo hypertrophy. A
shorter distance indicates faster hypertrophy. As expected, treatment of
wild-type limbs with Shh resulted in delayed chondrocyte hypertrophy, as shown
by an increased distance between the articular end and the
Ihh/Col10a1 expression domains
(Fig. 1A,B). In the cultured
PTHrP-/- mouse limb, chondrocyte hypertrophy was
accelerated when compared with the wild-type control
(Fig. 1A,B). However, treatment
of PTHrP-/- limb explants with Shh further accelerated
chondrocyte hypertrophy, when compared with the untreated
PTHrP-/- limb cultures
(Fig. 1A,B). Furthermore,
treatment of the PTHrP-/- limb explants with the Hh
inhibitor cyclopamine significantly delayed chondrocyte hypertrophy and
increased the domain of proliferating chondrocytes. These results indicate
that Ihh signaling promotes chondrocyte hypertrophy in the absence of
PTHrP.
Overexpression of Ihh in the cartilage in the absence of PTHrP accelerates chondrocyte differentiation in vivo
To rule out the possibility that the PTHrP-/- limb
explants may respond to Hh signaling differently in vitro, we analyzed the
PTHrP-independent role of Ihh signaling in vivo. We used a
UAS-Gal4 bigenic system to overexpress the chicken
Ihh gene in the cartilage (Yang
et al., 2003) and then crossed it with
PTHrP-/- mice to generate a compound mutant
UAS-cIhh;Col2a1-Gal4;PTHrP-/-. At E14.5,
PTHrP-/- mutant embryos showed accelerated hypertrophy
compared with wild-type embryos, as shown by the reduction of the distance
from the Col10a1 expression domain to the articular end
(Fig. 1C). In the
UAS-cIhh;Col2a1-Gal4 mutant embryos, chondrocyte hypertrophy
indicated by Col10a1 expression was slightly accelerated compared
with that of the wild type (Fig.
1C). Interestingly, in the
UAS-cIhh;Col2a1-Gal4;PTHrP-/- compound mutant embryos, the
proliferating chondrocyte region before hypertrophy was significantly shorter
in length even than that of the PTHrP-/- mutant embryos
(Fig. 1C). These results
indicate that in the absence of PTHrP signaling, upregulation of Ihh signaling
accelerates chondrocyte hypertrophy at an early stage of long bone development
in vivo.
|
). As shown
before, we found that in the Ptch1c/-;Col2a1-Cre embryos,
Ihh signaling is upregulated, as indicated by the enhanced expression of the
Hh signaling target genes Gli1 and Hip1
(Fig. 2B). In addition,
chondrocyte hypertrophy was significantly delayed, as there was no
Col10a1 expression at E14.5 when it was already strongly expressed in
wild-type and PTHrP-/- mutant embryos
(Fig. 2B). The delay of
chondrocyte hypertrophy is due to the upregulation of PTHrP
expression (Mak et al., 2006).
Interestingly, when we crossed the Ptch1c/-;Col2a1-Cre
mutant to the PTHrP-/- mutant, we found that ossification
and mineralization, as indicated by Alizarin Red staining, were strongly
enhanced in PTHrP-/-;Ptch1c/-;Col2a1-Cre double
mutants when compared with both Ptch1c/-;Col2a1-Cre and
the PTHrP-/- single mutant alone
(Fig. 2A). When sections of the
humerus at E14.5 were examined, we found that chondrocyte hypertrophy was
accelerated more than that of the PTHrP-/- embryos. There
were a few rows of columnar chondrocytes in the PTHrP-/-
embryo, but in the PTHrP-/-;Ptch1c/-;Col2a1-Cre
double mutant loss of columnar chondrocytes was so fast that there were few
columnar chondrocytes left, and the size of the articular cartilage region was
also reduced owing to accelerated articular chondrocyte differentiation
(Fig. 2B). In addition, the
Col10a1 expression domain in the
PTHrP-/-;Ptch1c/-;Col2a1-Cre double mutant was
closer to the articular end than that in the PTHrP-/-
embryo (Fig. 2B). Furthermore,
we performed a BrdU incorporation assay, which shows the percentage of cells
in the S phase of mitotic division and allows the comparison of cell
proliferation. In the PTHrP-/- embryo, there were still a
few BrdU-positive columnar chondrocytes
(Fig. 3A). Consistent with the
accelerated loss of the fast proliferating columnar chondrocytes in the
PTHrP-/-;Ptch1c/-;Col2a1-Cre double mutant,
there were hardly any BrdU-positive cells outside of the periarticular region
(Fig. 3A). Cell proliferation
in the columnar region was similarly reduced in the
PTHrP-/- and
PTHrP-/-;Ptch1c/-;Col2a1-Cre double mutant
(Fig. 3B), supporting the
previous notion that PTHrP signaling provides the competence of chondrocytes
to respond to Ihh signaling to promote proliferation
(Karp et al., 2000). These
results demonstrate that upregulation of Ihh signaling in chondrocytes
promotes chondrocyte hypertrophy independently of PTHrP.
Inhibition of Hh signaling delays chondrocyte hypertrophy independently of PTHrP
Our finding that upregulated Hh signaling promotes chondrocyte hypertrophy
in the absence of PTHrP suggests that inhibition of Ihh signaling
would result in delayed chondrocyte hypertrophy in the absence of
PTHrP. To test this, we inactivated Hh signaling by removing
Smo in chondrocytes of PTHrP-/- mouse embryos and
examined the pace of chondrocyte hypertrophy. Skeletal preparations of E15.5
embryos showed that the general morphology of the
PTHrP-/-;Smoc/c;Col2a1-Cre double mutant was
similar to that of Smoc/c;Col2a1-Cre single mutant, but
that mineralization in the double mutant was decreased compared with that in
the wild-type control and the PTHrP-/- single mutant
(Fig. 4A). More detailed
analysis of the E15.5 tibia sections revealed that almost all chondrocytes in
the double mutant embryo still expressed Col2a1, a marker for
non-hypertrophic chondrocytes, whereas a significant portion of chondrocytes
in the PTHrP-/- single mutant embryos had lost
Col2a1 expression and undergone hypertrophy
(Fig. 4B). In addition, more
chondrocytes in the wild-type embryo had lost Col2a1 expression than
had those in the Smoc/c;Col2a1-Cre single mutant
(Fig. 4B). Consistent with the
expression of Col2a1, the expression domains of Col10a1 and
the regions between the two Ihh expression domains were smaller in
Smoc/c;Col2a1-Cre and
PTHrP-/-;Smoc/c;Col2a1-Cre mutant embryos than
in wild-type and PTHrP-/- embryos, respectively
(Fig. 4B). These data indicate
that the progression of chondrocyte hypertrophy is slower when Smo is
removed, especially in the absence of PTHrP at early stages of
cartilage development. Therefore, in contrast to the role of PTHrP
that is activated and maintained by Ihh signaling, Ihh signaling is required
to promote chondrocyte hypertrophy in the absence of PTHrP. This
agrees with, and explains, the observation that in the
Ihh-/- embryo there is an initial delay of chondrocyte
hypertrophy at E14.5 (St-Jacques et al.,
1999).
|
), raising
the possibility that the loss of Wnt/β-catenin signaling actually
diminished the role of Ihh signaling in promoting chondrocyte hypertrophy. To
address this, we performed in situ hybridization with Lef1, which is
a transcriptional target of Wnt/β-catenin signaling
(Hovanes et al., 2001), on
E14.5 limb sections of Ptch1c/-;Col2a1-Cre mutants
(Fig. 5A). Lef1 is
mainly expressed in proliferating chondrocytes
(Fig. 5). In the
Ptch1c/-;Col2a1-Cre mutants, Lef1 expression was
highly upregulated in all chondrocytes, suggesting that Ihh signaling
activates Wnt/β-catenin signaling in proliferating chondrocytes. To
further confirm the effect of Hh signaling on Wnt/β-catenin signaling, we
isolated primary chondrocytes from rib cages of postnatal day one (P1)
wild-type mice. The Wnt/β-catenin signaling activity in the cultured
primary chondrocytes was measured by the TOPFLASH luciferase activity
(Korinek et al., 1997), which
is a reporter containing luciferase driven by Lef/Tcf binding sites and a
basic promoter. TOPFLASH luciferase expression is activated by β-catenin
when it binds Lef/Tcf factors. Consistent with the in vivo observation,
activation of the TOPFLASH reporter was detected in the
Cre-adenovirus-infected Ptch1c/c primary chondrocytes and
in the wild-type primary chondrocytes treated with Shh, when compared with
control cells (Fig. 5B,C),
indicating that Wnt/β-catenin signaling is activated by Hh signaling. In
addition, by quantitative RT-PCR, we found that the expression of two
canonical Wnt target genes, Axin2 and Lef1, was
significantly increased in the Shh-treated or Ptch1-deficient
chondrocytes when compared with the control cells
(Fig. 5D,E). By contrast, the
upregulation of canonical Wnt signaling activity in response to Hh signaling
was abolished when the primarily chondrocytes were treated with Dkk1, a
secreted antagonist of the canonical Wnt signaling
(Fig. 5C,E). Taken together,
our results suggest that upregulated Ihh signaling activates the expression of
some Wnt ligands that signal through the canonical Wnt pathway in
chondrocytes. Indeed, we found that the expression of several Wnt genes,
including Wnt2, Wnt3a, Wnt8 and Wnt8b, that can signal
through the canonical pathway was upregulated by activated Hh signaling (data
not shown). Furthermore, consistent with the previously observed upregulation
of Bmp expression in the developing cartilage of
Ptch1c/-;Col2a1-Cre mutant embryos
(Mak et al., 2006), we found
that Bmp signaling activity was upregulated in
Ptch1c/-;Col2a1-Cre mutant embryos, as indicated by the
increased phosphorylation of Smad1 in the chondrocytes
(Fig. 5F,G).
|
). To
test this, we activated Hh signaling in postnatal cartilage using the floxed
Ptch1 allele and an inducible chondrocyte-specific Cre mouse
line, Col2a1-CreER (Maeda et al.,
2007; Nakamura et al.,
2006). Cre recombinase activity was induced in this mouse line by
tamoxifen (TM). Skeletal preparations of P15
Ptch1c/c;Col2a1-CreER mice showed a significant
acceleration of mineralization, as indicated by increased Alizarin Red
staining in both forelimbs and hindlimbs. Conversely, the cartilage region
stained by Alcian Blue at the joint was significantly reduced
(Fig. 6A). Histological
sections of the distal femur showed that chondrocyte hypertrophy around the
secondary ossification center in Ptch1c/c;Col2a1-CreER
mice was greatly enhanced when compared with the wild-type control
(Fig. 6B, parts a,b). In
addition, immunohistochemistry with Col2a1 antibodies confirmed that the zone
of hypertrophic chondrocytes was larger with lower Col2a1 expression in
Ptch1c/c;Col2a1-CreER mice
(Fig. 6B, parts c,d).
Furthermore, in TM-induced one-year-old
Ptch1c/c;Col2a1-CreER mice, the number of chondrocytes
lining the joint surface and the proteoglycan content in articular
chondrocytes, as indicated by Hematoxylin/Eosin or Safranin O staining, were
reduced (Fig. 6B, parts e-j).
There was no obvious change in the growth plates in TM-induced
Ptch1c/c;Col2a1-CreER mice (see Fig. S2 in the
supplementary material). Conversely, in 14-month-old
Smoc/c;Col2a1-CreER mice, chondrocytes lining the joint
surface and the proteoglycan content in articular chondrocytes were increased
(Fig. 6C). These data indicate
that Hh signaling in postnatal cartilage promotes chondrocyte hypertrophy in
the area of secondary ossification. These results further suggest that
activated Hh signaling might be a risk factor for osteoarthritis.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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The negative-feedback loop of Ihh-PTHrP signaling plays a crucial role in
controlling the pace of chondrocyte hypertrophy
(Lanske et al., 1996;
St-Jacques et al., 1999;
Vortkamp et al., 1996).
However, Ihh signaling has also been found to promote chondrocyte
proliferation and the transition from slowly proliferating periarticular
chondrocytes to fast proliferating columnar chondrocytes (periarticular
chondrocyte differentiation) independently of PTHrP signaling
(Kobayashi et al., 2005b). Our
data uncovered another PTHrP-independent activity of Ihh in promoting
chondrocyte hypertrophy that was masked by the more potent
PTHrP-dependent activity of Ihh, which is to inhibit chondrocyte
hypertrophy in embryonic development (Fig.
7). These results highlight the highly cell context-dependent
effects of Ihh signaling. Ihh signaling promotes the proliferation of
periarticular and columnar chondrocytes, whereas, in more mature
prehypertrophic chondrocytes, it may promote hypertrophy by promoting cell
cycle exit.
|
), the
proliferating columnar chondrocyte region would be increased if chondrocyte
hypertrophy were not altered. Our observation that the columnar chondrocyte
region was shorter in the PTHrP-/-;Ptch1c/-;
Col2a1-Cre double mutant than in the PTHrP-/- single
mutant (Fig. 2B,
Fig. 3A) demonstrates that Hh
signaling also acts to promote chondrocyte hypertrophy in the absence of
PTHrP to reduce columnar chondrocyte number. When Ihh was
overexpressed while PTHrP signaling was maintained at a constant level in the
caPPR;PTHrP-/-;Ihh-Bg mutant, the periarticular
chondrocyte region was smaller but the length of the columnar region was
expanded significantly (Kobayashi et al.,
2005b). In this case, upregulated Ihh signaling promotes
periarticular chondrocyte differentiation and chondrocyte proliferation, and
both activities increase the number of proliferating columnar chondrocytes.
Although chondrocyte hypertrophy is also accelerated in this case, it is not
enough to cancel out the effects of enhanced periarticular chondrocyte
differentiation and chondrocyte proliferation. Furthermore, it has been shown
that PTHrP signaling is required to provide a competent domain in which Ihh
signaling can promote chondrocyte proliferation
(Karp et al., 2000). Therefore,
the difference between the
PTHrP-/-;Ptch1c/-;Col2a1-Cre double mutant and
the caPPR;PTHrP-/-;Ihh-Bg mutant is that chondrocyte
proliferation is greatly reduced in the
PTHrP-/-;Ptch1c/-;Col2a1-Cre double mutant.
Hence, the significantly increased columnar chondrocyte region in the
caPPR;PTHrP-/-;Ihh-Bg mutant is a result of increased
chondrocyte proliferation. These results also suggest that the
PTHrP-independent role of Ihh signaling in promoting chondrocyte
hypertrophy may be stronger than that in promoting periarticular
differentiation in the embryonic cartilage. Overall, the
PTHrP-independent function of Ihh signaling is to increase the
transition rate through the different chondrocyte zones during endochondral
bone formation.
One crucial factor that determines the relative strength of the opposite
activities of the PTHrP-dependent and -independent roles of Ihh
signaling in chondrocyte hypertrophy is the level of PTHrP
expression. PTHrP expression in the cartilage was progressively
weaker in older embryos (K.K.M. and Y.Y., unpublished) and is further reduced
in adult animals (Tsukazaki et al.,
1995). Therefore, when PTHrP signaling is robust in the embryonic
cartilage, upregulated Ihh signaling predominantly delays chondrocyte
hypertrophy by upregulating PTHrP expression. However, when mammals
approach adulthood, cartilage hypertrophy and bone formation accelerate
coordinately as longitudinal bone growth declines and the growth plate is
either reduced (i.e. in mouse) or closed (i.e. in human). This is consistent
with reduced PTHrP levels postnatally. It is also conceivable that when PTHrP
signaling is low enough, the PTHrP-independent role of Hh signaling
in promoting chondrocyte hypertrophy is significant for postnatal cartilage
development and homeostasis. Furthermore, because only periarticular cells and
the upper layer of articular chondrocytes are competent to express
PTHrP, even when Hh signaling is upregulated throughout the cartilage
(Mak et al., 2006), somatic
mutations that upregulate Hh signaling only in chondrocytes deep in the joint
cartilage and growth plates will not upregulate PTHrP expression.
Hence, when the ability to strongly express PTHrP is lost,
upregulated Ihh signaling, for instance due to mutations in Ptch1 (as
we have shown in the Ptch1c/c;Col2a1-CreER mutant), may be
a risk factor for osteoarthritis.
Our finding that Hh signaling activated Wnt/β-catenin and Bmp
signaling in chondrocytes has provided some insight into the underlying
mechanism of Hh signaling in promoting chondrocyte hypertrophy.
Wnt/β-catenin signaling has been shown to promote chondrocyte
hypertrophy, at least in part, through downregulating Sox9 protein levels
(Akiyama et al., 2004), a
transcription factor that is required for chondrocyte formation and also
inhibits chondrocyte hypertrophy (Bi et
al., 2001). However, Wnt/β-catenin signaling may not be the
only downstream target of Hh signaling in regulating chondrocyte hypertrophy.
Indeed, upregulated Hh signaling leads to many changes. For example, we have
found that the expression and signaling activity of Bmps are upregulated by
enhanced Hh signaling (Mak et al.,
2006) (this study), and Bmp signaling activity has been shown to
promote chondrocyte hypertrophy independently of PTHrP signaling
(Kobayashi et al., 2005a). It
is noted that, when Hh signaling is reduced by removing Ihh in postnatal
cartilage from P1, chondrocyte hypertophy is also accelerated, although
PTHrP expression is still detected
(Maeda et al., 2007). These
results suggest that Hh signaling still inhibits chondrocyte hypertrophy in
postnatal cartilage, and this difference can be reconciled by the following
considerations. First, Hh signaling was activated cell autonomously in the
Ptch1c/c;Col2a1-CreER mutant cartilage by injecting the
lactating female mouse at P3. Therefore, Hh signaling activation in our system
occurs later and PTHrP expression might be further declined at that
time. Second, the effects of Ihh signaling in chondrocyte hypertrophy can be
mediated by PTHrP, Bmp and Wnt signaling simultaneously. As PTHrP and Bmp and
Wnt signaling exhibit opposite effects in regulating chondrocyte hypertrophy,
when Hh signaling is perturbed (up- or downregulated) in the adult cartilage,
the outcome of chondrocyte hypertrophy depends on the relative strength of the
pro- and anti-hypertrophy signaling. It is likely that in
Ptch1c/c;Col2a1-CreER mice when Hh signaling is
upregulated after P3, the pro-hypertrophy signaling (i.e. by Bmps and Wnts) is
more robust than is the anti-hypertrophy signaling by PTHrP.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/11/1947/DC1
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ACKNOWLEDGMENTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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