1 Department of Metabolic Disorders, Amgen, One Amgen Center Drive, Thousand
Oaks, CA 91320, USA
2 Department of Cancer Biology, Amgen, One Amgen Center Drive, Thousand Oaks, CA
91320, USA
3 Department of Pathology, Amgen, One Amgen Center Drive, Thousand Oaks, CA
91320, USA
4 Department of Protein Science, Amgen, One Amgen Center Drive, Thousand Oaks,
CA 91320, USA
5 Department of Stem Cell Regulation, Institute of Medical Science, The
University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639,
Japan
Author for correspondence (e-mail:
naoki.nakayama{at}petermac.org)
Accepted 2 October 2003
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SUMMARY |
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Key words: Secreted protein, Chordin, BMP, Inhibitor, Chondrocyte, Cartilage, Superficial zone, Joint, Osteoarthritis
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Introduction |
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The chordin polypeptide contains four CRs, of which the first and the third
(CR1 and CR3) are responsible for BMP binding
(Larrain et al., 2000).
Binding of chordin to BMP4 is specific and tight
(Piccolo et al., 1996
).
Proteolysis by Tolloid (or BMP1), which liberates CR1 and CR4 from chordin, is
required to release bound BMP4 (Piccolo et
al., 1997
; Scott et al.,
1999
). The importance of CR for BMP interactions has been
strengthened by the recent finding that connective tissue growth factor
functions as a BMP-binding inhibitor, and that its single CR domain is
essential for this activity (Abreu et al.,
2002
).
We previously described a small chordin-like secreted protein, CHL1 (for
chordin-like 1, re-designated from CHL), a novel BMP-binding inhibitor with
three CRs (Nakayama et al.,
2001). CHL1 was isolated originally from mouse bone marrow stromal
cells. Interestingly, CHL1 expression correlates with the
stem/progenitor-support activities of over 19 stromal cell lines established
from the aorta-gonadsmesonephros region, the site at which definitive
hematopoietic stem cells first arise during embryogenesis
(Oostendorp et al., 2002
).
However, CHL1 mRNA is also detected in various mesenchymal derivatives
associated with (1) the dermatome, limb bud and chondrocyte precursors of the
skeleton during embryogenesis, and (2) digestive tract connective tissues,
kidney tubules and marrow stromal cells in adults. In addition, CHL1
is expressed in olfactory bulb and cerebellum, suggesting a wider array of
physiological functions. Two other groups have independently isolated CHL1,
naming it neuralin-1 and ventroptin
(Coffinier et al., 2001
;
Sakuta et al., 2001
) and
demonstrating its ability to correctly specify retinotectal projections along
the dorsoventral retinal axis during development.
We provide evidence that CHL2, a novel chordin family member with
structural homology to CHL1, is a BMP-binding inhibitor whose expression is
uniquely restricted to the superficial layers of developing joint cartilage,
in contrast to that of other family members. Potential downregulation of
cartilage matrix accumulation and/or cartilage mineralization by CHL2 is
suggested by in vitro observations using cartilage particles derived from
embryonic stem (ES), cell-derived mesodermal cells
(Nakayama et al., 2003) and
with marrow-derived mesenchymal stem/progenitor cells (MSCs). CHL2 is also
induced in osteoarthritic joint cartilage, implying a potential role during
cartilage regeneration in the adult.
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Materials and methods |
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Isolation of mouse, rat and human CHL2 cDNAs
Mouse placentas were isolated at E18. A signal-trap cDNA library and a
regular full-length cDNA library were constructed as described previously
(Nakayama et al., 2001). From
400 trap-positive clones sequenced, a cDNA fragment encoding the
NH2-terminal sequence of a putative secreted protein with
significant homology to Xenopus chordin was identified (designated
mouse CHL2 or mCHL2) by a BLAST search (Accerlys, San Diego, CA). Using this
partial cDNA as a probe, the corresponding full-length cDNA (approximately 1.8
kb) was isolated, and designated as pSPORTmCHL2.
A human placenta library was constructed with size-selected (>1.5 kb) oligo(dT)-primed cDNAs in the pSPORT1 vector (Gibco). A full-length human cDNA clone (hCHL2, 1.5 kb in length) was isolated using the mouse cDNA probe, and designated pSPORThCHL2. Rat CHL2 (rCHL2) cDNA was cloned by PCR from a rat fetal liver cDNA library (Stratagene) using the following primers: sense, 5'-TCCTCTCATCCTCACCTTAG-3' (based on mCHL2-5'UTR), and antisense, 5'-GAGGGTAATGCGACTTCTTT-3' (based on mCHL2-3'UTR). A 1.2 kb fragment was amplified using Advantage-HF2 enzyme (Clontech), cloned into pTOPO2.1 (Invitrogen), and designated pTOPOrCHL2.
Production, purification, and detection of recombinant CHL2 protein
To prepare mCHL2, the mCHL2 open reading frame (ORF) was mutated
by PCR to replace the stop codon with a SalI site, inserted into a
pFLAG-CMV5a expression vector (Sigma) to attach the FLAG sequence in-frame to
mCHL2 at its COOH terminus (mCHL2-FLAG), and designated pFLAGmCHL2. Expression
was checked by transient transfection of 293T cells, followed by direct
western blot analysis of conditioned media, using the anti-FLAG monoclonal
antibody M2 as described previously
(Nakayama et al., 2001).
A large-scale, transient transfection-based expression was performed as
described with 293T cells bearing pFLAGmCHL2
(Nakayama et al., 2001),
yielding approximately 10 µg/ml of mCHL2-FLAG. The protein was purified by
affinity chromatography using anti-FLAG M2 affinity gel (Sigma) under
high-salt conditions, as described by Piccolo et al.
(Piccolo et al., 1997
), after
which positive fractions were subjected to hydroxyapatite column
chromatography (equilibrated with 10 mM phosphate, with gradient elution from
10 mM to 400 mM phosphate) at pH 6.9. Purity was confirmed by
SDS-polyacrylamide gel electrophoresis followed by Coomassie Blue staining.
Approximately 5 mg of >95% pure mCHL2-FLAG protein were obtained from 2.5 l
of conditioned medium.
A rabbit polyclonal antibody for mCHL2 (CHL2-COOH) was raised to the
peptide NH2-CPEDEAEDDHSEVISTR-COOH, and affinity purified against
the corresponding peptide (Harlow and
Lane, 1988
).
Co-immunoprecipitation analysis
Immunoprecipitations to demonstrate direct interactions between BMPs,
TGFßs and activin A were performed as described previously
(Nakayama et al., 2001) except
that only one condition was used: 200 ng mCHL2-FLAG were mixed with 100 ng of
BMP/GDF/activin/TGFß in 1 ml binding buffer, followed by 12 µg/ml of
CHL2-COOH. The BMP/GDF/activin/TGFß immunocomplex was precipitated
with 20 µl protein A agarose beads (Santa Cruz), fractionated on an
SDS-polyacrylamide gel (NuPAGE, Invitrogen), blotted, and visualized with the
corresponding antibody as described previously
(Nakayama et al., 2001
), or
with 1 µg/ml of anti-BMP2, anti-BMP7, anti-TGFß1 or anti-TGFß3 or
0.3 µg/ml of anti-GDF5. Each blot then was treated with 4.4 µg/ml M2 to
confirm the precipitation of mCHL2-FLAG. The inhibitory effect of mCHL2-FLAG
(0.1-1 µg, in 1 ml binding buffer) on BMP4 binding (at 100 ng/ml) to the
BMPR1B ectodomain (BMPR1B-Fc at 1 µg/ml) was performed as described by
Nakayama et al. (Nakayama et al.,
2001
), except that BMP4 visualization on blots was followed by
CHL2 and BMPR1B-Fc detection using 4.4 µg/ml M2 and 2.2 µg/ml
anti-IgGFc, respectively.
Ectopic axis formation in the Xenopus embryo
Inhibition of BMP4 by mCHL2 was assessed in Xenopus embryos. The
EcoRI-NotI fragment of pSPORTmCHL2 was cloned into the
EcoRI-NotI sites of pCS2+
(Rupp et al., 1994), and the
resulting plasmid was linearized with NotI. Capped mRNAs were
synthesized with SP6 polymerase, quantified, diluted and injected into two
ventral blastomeres as described previously
(Nakayama et al., 2001
).
Alkaline phosphatase induction in C2C12 cells by BMPs
Promyoblast C2C12 cells were maintained and differentiated according to the
method of Kirsch et al. (Kirsch et al.,
2000a). Briefly, cells were plated at 3x104
cells/well in a 96-well plate, and after 1 day, stimulated to differentiate
for 72 hours in 120 µl of Dulbecco's Modified Eagle's medium with 2% calf
serum (Gibco) in the presence or absence of BMP and/or CHL2. Cells were washed
and lysed. Alkaline phosphatase (AP) activity was measured with
p-nitrophenyl phosphate (Sigma), with specific activity calculated as
the amount of p-nitrophenol produced in 30 minutes at 37°C,
normalized to total protein content, as determined with BCA reagent
(Pierce).
In situ hybridization and northern blot analysis
Northern blotting was performed against the entire open reading frame (ORF)
of CHL2 on human and mouse multiple tissue RNA blots (Clontech) using
the 32P-labeled XbaI-SalI fragment from
pSPORThCHL2 as a probe (Sambrook et al.,
1989).
In situ hybridization (ISH) was performed on 5 µm paraffin sections of
zinc formalin-fixed, formic acid-decalcified tissue according to the method of
Wilcox (Wilcox, 1993),
including a high stringency wash with 0.1x SSC at 55°C. Slides
hybridized with 33P-labeled RNA probes were exposed to NTB2
emulsion (Kodak) for 3 weeks, developed and counterstained with Hematoxylin
and Eosin. Expression was subjectively evaluated under dark-field
microscopy.
For mCHL2, the 1234-bp SalI-NotI fragment and the 653 bp HindIII fragment were deleted from pSPORTmCHL2 to obtain pSPmCHL2-COOH and pSPmCHL2-NH2, respectively. Both plasmids were linearized with EcoRI to generate non-overlapping probes. An hCHL2-bearing plasmid, pSPhCHL2NH2, was made by deleting the 811 bp BamHI fragment from pSPORThCHL2, leaving the 746 bp hCHL2 NH2-terminal ORF. The plasmid was linearized with SalI. RNA probes for hCHL2 and mCHL2 were synthesized with SP6 RNA polymerase. For rCHL2, an approximately 1.2 kb fragment of rCHL2 cDNA derived from pTOPOrCHL2 was cloned into the pBluescript vector (pBSrCHL2). The plasmid was linearized with XbaI and a labeled RNA probe synthesized with T7 RNA polymerase.
CHL2 expression was assessed by ISH in mouse embryonic/adult
tissues, and in structurally normal and arthritic joints of adult humans (knee
cartilage) and rats (hind paws). Human diseased cartilage samples from
rheumatoid arthritis (RA) or osteoarthritis (OA)/degenerative joint disease
(DJD) patients were provided by Cooperative Human Tissue Network, and normal
control samples were from Zoion Diagnostics (New York, NY), with pre-approval
of the Institutional Review Board (758WIRB and CP1098/01, respectively).
Collagen-induced arthritis (CIA) was induced, as described
(Trentham et al., 1977), in
anesthetized female Lewis rats (7-8 weeks old, 150-170 g) by intradermal
injection of porcine COL2 (Chrondex, Seattle, WA) emulsified 1:1 in incomplete
Freund's adjuvant (Difco, Detroit, MI) at 10 different sites over the back (50
µg COL2/100 µl/injection). Disease developed between 10 and 12 days
after injection, as determined by caliper measurements (Cole Parmer, Vernon
Hills, IL) of ankle width and ambulatory difficulties. Paws were harvested for
ISH 7 days after CIA onset. These experiments were conducted in accordance
with federal animal care guidelines and were pre-approved by the Amgen
Institutional Animal Care and Use Committee.
Chondrogenic differentiation of MSCs and gene expression analysis
Human MSCs were cultured and differentiated as described
(Mackay et al., 1998).
Briefly, the pellet culture was performed in serum-free chondrogenesis medium
supplemented with 10 ng/ml TGFß3, with or without 2 µg/ml mCHL2-FLAG,
1 µg/ml noggin-Fc, or 1 µg/ml IgGFc. On days 21-28, cartilage-like
particles were formalin-fixed, paraffin-embedded, sectioned centrally and
stained with Toluidine Blue to detect sulfated glycosaminoglycans
(Nakayama et al., 2003
;
Sheehan and Hrapchak, 1987
).
Three sections from different regions were examined to confirm staining
reproducibility.
To analyze gene expression, two to five particles were harvested at
designated times and disrupted immediately in guanidine isothiocyanate
solution (RNeasy kit, Qiagen). Total RNA was purified using the manufacturer's
protocol, including DNase I treatment. Reverse transcription (RT) and PCR were
performed as described previously
(Nakayama et al., 1998),
except that the PCR used 30 cycles, an annealing temperature of 62°C and
one primer set per gene. Primers for aggrecan, cartilage oligomeric matrix
protein (COMP), COL1, COL2, COL10, SOX9, CHL1, CHL2 and
glyceraldehyde-3-phosphate dehydrogenase (GAPD) are shown in
Table 1.
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Results |
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MCHL2 induced a secondary axis in the Xenopus embryo
Chordin is known to dorsalize the gastrulating Xenopus embryo by
inhibiting BMP4 activity, so the impact of CHL2 on Xenopus embryo
development was examined (Table
2). Injection of 1 pg mCHL2 RNA per blastomere induced trunk
duplication in 74% of embryos, compared with 0% for uninjected controls and
embryos given EF1 mRNA. As a positive control, injection of 10 pg
mCHL1(s2) RNA yielded an axis duplication rate of 80%
(Nakayama et al., 2001
). These
results indicated that mCHL2 actively antagonized an endogenous ventralizing
factor (presumably BMP4). The improved efficacy afforded by a 10-fold lower
CHL2 dose suggested that it might be a more stable and/or potent BMP inhibitor
than CHL1.
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|
Chordin and CHL1 inhibit BMP activity by blocking their interactions with their receptors. Therefore, we determined whether CHL2 had a similar function by mixing an Fc-fusion protein, incorporating the extracellular domain of BMP receptor 1B (BMPR1B-Fc), with BMP4 and mCHL2-FLAG, followed by precipitation of the BMPR1B-Fc complex with protein A beads. As shown in Fig. 2C, BMP4 co-precipitated specifically with BMPR1B-Fc, but not IgG-Fc, in the absence of mCHL2-FLAG. However, the signal for co-precipitated BMP4 weakened appreciably as increasing amounts of mCHL2-FLAG were added (particularly at 0.3 µg/ml or higher). Co-precipitation of mCHL2-FLAG was not detected. These results suggest that CHL2 acts like chordin and CHL1 to prevent BMP4 interacting with its receptor.
CHL2 inhibits BMP in vitro
Next, we demonstrated that the recombinant mCHL2 inhibited BMP activity by
quantifying BMP-dependent AP induction in C2C12 cells. A serially diluted BMP
inhibitor [chordin (mCHD-His), mCHL2-FLAG, or noggin-Fc] was mixed with BMP2,
BMP4, BMP6 or BMP7 at a concentration corresponding to the EC50 for AP
induction in C2C12 cells and then cultured for 3 days. Cell-bound AP activity
was then measured (Fig. 3,
Table 3). CHL2 inhibited AP
induction by all four BMPs. Noggin-Fc and mCHL2-FLAG reproducibly elicited
similar, dose-dependent inhibitions of BMP4, with complete suppression
occurring at concentrations of 1-3 µg/ml (20-60 nM). The mCHD-His activity
was weakest for BMP4; it was approximately fivefold less potent than
mCHL2-FLAG. Partially purified mCHL1(s2)-FLAG and mCHD-FLAG
(Nakayama et al., 2001)
inhibited BMP4 with a potency indistinguishable from that of mCHD-His (not
shown). In contrast, both noggin-Fc and mCHD-His displayed weaker inhibitory
activities than mCHL2-FLAG toward BMP6 and BMP7. In particular, noggin-Fc was
approximately sevenfold less potent on BMP6 than mCHL2-FLAG. Thus, CHL2
inhibits BMP2, BMP4, BMP6 and BMP7 as well as or better than noggin and
chordin.
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In mouse reproductive organs, ISH detected CHL2-positive connective tissues
such as ligaments of the ovary and oviduct in females, and of testis,
epididymis and certain male accessory sex glands in males. Expression was high
in uterine myometrium (Fig.
5Ba), as was that of CHL1 (not shown). CHL2 was also present in
maternally derived placental tissues (not shown). A trace CHL2 signal was
found on colonic serosa (Fig.
5Bb); in contrast, CHL1-positive cells lie between the colonic
submucosa and muscularis (Nakayama et al.,
2001). Interestingly, CHL1 but not CHL2 was
expressed in stomach and small intestine
(Nakayama et al., 2001
). In
rat tissues, CHL2 occurred at low to moderate levels in cervical muscles and
discrete regions of the placenta (not shown).
CHL2 mRNA expression in diseased cartilage
Degenerating cartilage from human arthritis patients and rats with CIA were
assessed by ISH (Fig. 6). In
two relatively normal specimens from knees of adult humans, CHL2 mRNA was
expressed in a few chondrocytes in the superficial zone as well as in the
middle zone (Fig. 6A). In 19 OA
cases, expression was limited to chondrocytes in the middle zone, where
numerous well-labeled cells were observed
(Fig. 6B,C); positive cells
were not found in the superficial zone in any OA sample. Interestingly, 50-90%
of such CHL2-expressing chondrocytes existed in clusters of 2-3
cells. Unlike OA, two RA specimens exhibited weak expression in both the
superficial and middle zones (Fig.
6D). As with humans, scattered chondrocytes in normal articular
cartilage of rats expressed CHL2
(Fig. 6E), while CIA paw joints
had similar (Fig. 6F) or fewer
labeled chondrocytes relative to controls.
In summary, CHL2 was expressed in normal and diseased cartilage in humans and rats. It was expressed most strongly in human OA patients, although the signal had shifted to the middle zone. Interestingly, CHL2 expression levels and patterns were not significantly altered, relative to normal cartilage, in the rat CIA model and human patients with RA.
Effects of CHL2 on MSC differentiation into chondrocytes
We further addressed the relevance of CHL2 to cartilage formation using
MSC, which can differentiate into chondrocytes in vitro
(Mackay et al., 1998) in
association with upregulated BMP transcription
(Roh et al., 2001
). As shown
by RT-PCR in Fig. 7A, hCHL2
mRNA, but not COL2 mRNA, was expressed by undifferentiated MSCs. Transcripts
for SOX9, COL1 and COL10 (not shown) as well as CHL1, aggrecan and COMP were
also detected. The CHL2 signal fell as chondrogenesis progressed; those for
SOX9 and COL10 (not shown) as well as CHL1, aggrecan and COMP
(Fig. 7A) maintained a similar
level throughout the culture period. In contrast, COL2 mRNA was tightly
regulated, with production induced by day 7 in culture conditions favoring
chondrogenesis but absent under osteogenic conditions (not shown)
(Jaiswal et al., 1997
).
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Effects of CHL2 on chondrocyte maturation
Chondrocytes from OA joints express markers of hypertrophy, such as COL10
and AP (Kirsch et al., 2000b;
Von der Mark et al., 1992
), so
we addressed whether CHL2 induction in OA cartilage would affect
differentiation and mineralization of hypertrophic chondrocytes. We
demonstrated previously that mesodermal progenitor cells, purified from
differentiating ES cells, can form hyaline cartilage particles in vitro that
will undergo further mineralization
(Nakayama et al., 2003
). We
isolated FLK1 PDGFR
+ mesodermal cells,
subjected them to pellet micromass culture, and induced cartilage matrix
mineralization (verified by von Kossa staining) in the presence or absence of
mCHL2-FLAG, noggin-Fc or BMP6 (Fig.
7C, Table 5).
Addition of 3 µg/ml mCHL2-FLAG significantly reduced the von Kossa-positive
matrix area in 75% of particles examined, of which half showed near-complete
inhibition. In contrast, COL10 expression was reduced slightly, while no
significant change was detected in COL2
(Fig. 7C). Noggin-Fc at 2-3
µg/ml provided similar, but somewhat weaker, inhibition. A positive in
vitro role for BMP6 has been suggested in chondrocyte hypertrophic
differentiation (Grimsrud et al.,
1999
). However, BMP6 at 50 ng/ml, which was sufficient to enlarge
the particle size, did not affect the degree of mineralization.
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Discussion |
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Structure and function of CHL2
Searches of human and mouse genome databases indicated that CHL2 is most
homologous to CHL1. Injection of CHL2 RNA induced trunk duplication in early
Xenopus embryos similar to those produced by chordin and CHL1(s2)
RNAs (Table 2)
(Nakayama et al., 2001).
Recombinant mCHL2 protein interacted directly with five BMPs and one GDF
(Fig. 2 and
Nakayama et al., 2001
) thereby
inhibiting, in vitro, several BMP/GDF-dependent processes including,
osteogenic differentiation of C2C12 mesenchymal progenitor cells by several
BMPs (Fig. 3,
Table 3), ATDC5 embryonal
carcinoma cells by GDF5 (not shown) and BMP4-dependent lymphohematopoietic
(CD34+CD31hi and CD34+CD31lo)
progenitor cell development from ES cells (not shown)
(Nakayama et al., 2000
). Under
our conditions, CHL2 provided 50% inhibition (IC50) by blocking a half to a
third of available BMP dimers, suggesting that tight CHL2 binding to one BMP
subunit might be sufficient for full inhibition. Furthermore, as with related
factors (chordin, noggin, CHL1), CHL2 prevented BMP interactions with the BMP
receptor (Fig. 2C), although
CHL2 activity was two- to sevenfold more potent than chordin
(Table 3) and CHL1 (not shown).
Thus, CHL2 is structurally and functionally similar to chordin and CHL1.
Potential roles of CHL2 in joint formation
Cartilages within hip and knee joints and at the costochondral junction
were the major CHL2 expression sites during embryogenesis
(Fig. 4). CHL2 mRNA was also
expressed strongly in connective tissues anchoring reproductive organs
(Fig. 5). CHL2 in developing
joints was restricted to superficial zone chondrocytes; expression was
substantially diminished in adult joint cartilage
(Fig. 4). The CHL2-expressing
areas did not overlap domains expressing chordin (non-chondrogenic mesenchyme
of limb buds), CHL1 (condensing mesoderm, hypertrophic chondrocytes) and
gremlin (non-chondrogenic regions of limb buds, including interdigital
mesenchyme) during limb formation
(Nakayama et al., 2001;
Scott et al., 1999
;
Scott et al., 2000
),
suggesting that these four factors have divergent biological roles. However,
as with CHL1 (Nakayama et al.,
2001
), CHL2 expression in developing cartilage overlapped with
noggin expression (Brunet et al.,
1998
; Capdevila and Johnson,
1998
; Merino et al.,
1998
; Nifuji and Noda,
1999
; Pathi et al.,
1999
).
As CHL2 is a BMP-binding inhibitor, and BMPs regulate multiple steps during chondrogenesis, expression of CHL2 in superficial chondrocytes in developing joints suggests a role in joint specification. The ability of exogenous mCHL2 to inhibit chondrogenesis by hMSCs supports this hypothesis (Fig. 7A,B, Table 4). The surface of developing cartilage consists of proliferating mesenchymal cell layers that are differentiating into chondrocytes. By its location, CHL2 might act as an important boundary in joint formation. A possible role could be to prevent articular cartilage from becoming too massive, by keeping mesenchymal cells in the joint space from being recruited to the chondrocyte developmental pathway.
Alternatively, CHL2 could play more subtle roles. The superficial zone of
articular cartilage is composed of flattened chondrocytes separated by
tangential arrays of thin collagen fibrils, but no proteoglycan matrix. In
contrast, the middle zone consists of rounded chondrocytes surrounded by a
proteoglycan-rich matrix containing radial bundles of thick collagen fibrils.
Osteogenic BMPs accumulate in the pericellular matrix of articular cartilage,
with highest levels in the middle to deep zone
(Anderson et al., 2000).
Conversely, the osteogenic antagonist BMP3
(Daluiski et al., 2001
) is more
highly expressed in the superficial zone. We failed to detect an interaction
between CHL2 and BMP3 (not shown), suggesting that preferential expressions of
CHL2 and BMP3 in the surface chondrocytes act to regulate a BMP gradient in
normal articular cartilage.
Potential involvement of CHL2 in osteoarthritis
CHL2 mRNA was never detected in the growth plate, where proliferation and
hypertrophic differentiation of prehypertrophic chondrocytes normally occur,
implying that CHL2 is not relevant to normal pathways of chondrocyte
proliferation and maturation. However, the up-regulation of CHL2 transcripts
specifically in middle zone cartilage of adult joints with OA
(Fig. 6) prompted our
speculation that CHL2 has a role in cartilage repair. We examined the
associations between CHL2 and three principal phenotypes of OA cartilage: (1)
reduced proteoglycan levels (which precede overt histological changes), (2)
aberrant chondrocyte proliferation (resulting in clonal chondrocyte
expansion), and (3) upregulation of molecules (e.g., COL1, COL3, COL10, and
AP) found in hypertrophied or de-differentiated chondrocytes but not normal
articular chondrocytes (Aigner et al.,
1993; Kirsch et al.,
2000b
; Von der Mark et al.,
1992
). First, the proteoglycan content in CHL2-expressing regions
of OA cartilage was not reduced, as detected in Toluidine Blue-stained
sections by the retained metachromasia (not shown). Second, CHL2-expressing
chondrocytes in OA cartilage were typically found as aggregates; however,
middle to deep zone chondrocytes are normally arranged in a cylindrical
fashion, so this association might reflect normal middle zone anatomy. In
contrast, the weak but significant inhibition of cartilage mineralization by
CHL2 (Fig. 7C,
Table 5) suggested that in OA
cartilages this molecule might delay and/or reduce the degree of chondrocyte
hypertrophy, thereby ameliorating cartilage degeneration. Further support for
this premise is that medium turbidity, which indicates mineral deposition and
excess Ca2+ excretion, was delayed during hypertrophic
differentiation culture of cartilage particles by CHL2 or noggin (not shown).
However, we have not addressed whether CHL2 is involved in the
de-differentiation of mature articular chondrocytes. Co-localization analyses
between cells expressing the CHL2 mRNA and those expressing transcripts for
COL1, COL10 or proliferating cell-nuclear antigen are underway to answer this
question.
In conclusion, abundant evidence suggests that BMP functions are regulated by numerous extracellular BMP-binding proteins in developing joints. Our current data support this paradigm and add a new BMP inhibitor, CHL2, to this pathway. Our findings also provide the first evidence that a chordin-like BMP-binding inhibitor might be intimately involved in the pathogenesis of degenerative joint disease.
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ACKNOWLEDGMENTS |
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Footnotes |
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